Compositions for biological systems and methods for preparing and using the same

ABSTRACT

A composition for influencing biological growth including live cells, a fluid comprising dextrose, a protectant, and a first cytokine having a first concentration within the composition as described within the present disclosure.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 15/619,467, filed on Jun. 10, 2017, which claims the benefit ofpriority to U.S. Provisional App. No. 62/349,633, filed on Jun. 13,2016, both of which are incorporated by reference in their entiretyherein for all purposes. Priority is claimed pursuant to 35 U.S.C. § 120and 35 U.S.C. § 119.

BACKGROUND OF THE INVENTION

The field of the invention generally relates to cellular allograft fortissue redevelopment, including bone redevelopment. The sources ofcellular allograft compositions have historically involved adult bonemarrow, though recently, placental fluid has been an experimentalsource.

SUMMARY OF THE INVENTION

In one embodiment of the present disclosure, a composition forinfluencing biological growth includes live cells, a fluid comprisingdextrose, a protectant, and a first cytokine having a firstconcentration within the composition, selected from the group consistingof: BDNF at between 492 picograms per ml and 915 picograms per ml, bNGFat between 14 picograms per ml and 26 picograms per ml, EGF at between715 picograms per ml and 1329 picograms per ml, Eotaxin at between 37picograms per ml and 69 picograms per ml, FGF-2 at between 28 picogramsper ml and 53 picograms per ml, GM-CSF at between 7 picograms per ml and15 picograms per ml, Gro-α at between 2974 picograms per ml and 5524picograms per ml, HGF at between 352 picograms per ml and 656 picogramsper ml, IFN-α at between 253 picograms per ml and 472 picograms per ml,IFN-γ at between 33 picograms per ml and 63 picograms per ml, IL-10 atbetween 67 picograms per ml and 126 picograms per ml, IL-12p70 atbetween 9 picograms per ml and 19 picograms per ml, IL-13 at between 11picograms per ml and 22 picograms per ml, IL-15 at between 30 picogramsper ml and 57 picograms per ml, IL-17A at between 19 picograms per mland 37 picograms per ml, IL-18 at between 78 picograms per ml and 146picograms per ml, IL-1α at between 712 picograms per ml and 1324picograms per ml, IL-1β at between 14 picograms per ml and 28 picogramsper ml, IL-1RA at between 2215 picograms per ml and 4114 picograms perml, IL-2 at between 16 picograms per ml and 31 picograms per ml, IL-21at between 370 picograms per ml and 689 picograms per ml, IL-22 atbetween 16 picograms per ml and 32 picograms per ml, IL-23 at between201 picograms per ml and 376 picograms per ml, IL-27 at between 21picograms per ml and 40 picograms per ml, IL-31 at between 11 picogramsper ml and 22 picograms per ml, IL-4 at between 17 picograms per ml and33 picograms per ml, IL-5 at between 15 picograms per ml and 28picograms per ml, IL-6 at between 50 picograms per ml and 95 picogramsper ml, IL-7 at between 26 picograms per ml and 50 picograms per ml,IL-8 at between 4504 picograms per ml and 8366 picograms per ml, IL-9 atbetween 131 picograms per ml and 245 picograms per ml, IP-10 at between469 picograms per ml and 873 picograms per ml, LIF at between 41picograms per ml and 77 picograms per ml, MCP-1 at between 14,480picograms per ml and 26,893 picograms per ml, MIP-1α at between 303picograms per ml and 565 picograms per ml, MIP-1β at between 375picograms per ml and 698 picograms per ml, PDGF-BB at between 807picograms per ml and 1500 picograms per ml, PIGF-1 at between 743picograms per ml and 1381 picograms per ml, RANTES at between 3277picograms per ml and 6087 picograms per ml, SCF at between 23 picogramsper ml and 45 picograms per ml, SDF-1α at between 1114 picograms per mland 2071 picograms per ml, TNF-α at between 42 picograms per ml and 80picograms per ml, TNF-β at between 12 picograms per ml and 25 picogramsper ml, VEGF-A at between 481 picograms per ml and 895 picograms per ml,and VEGF-D at between 46 picograms per ml and 88 picograms per ml.

In another embodiment of the present disclosure, a composition forinfluencing biological growth includes live cells, a fluid comprisingdextrose, a protectant, VEGF-A, at a concentration within thecomposition of between 481 picograms per ml and 895 picograms per ml,PDGF-BB, at a concentration within the composition of between 807picograms per ml and 1500 picograms per ml, EGF, at a concentrationwithin the composition of between 715 picograms per ml and 1329picograms per ml, SCF, at a concentration within the composition ofbetween 23 picograms per ml and 45 picograms per ml, and IL-1RA, at aconcentration within the composition of between 2215 picograms per mland 4114 picograms per ml.

In still another embodiment of the present disclosure, a composition forinfluencing biological growth includes live cells, a fluid comprisingdextrose, a protectant, and wherein the composition includes thefollowing cytokines with their concentration within the composition:BDNF at between 492 picograms per ml and 915 picograms per ml, bNGF atbetween 14 picograms per ml and 26 picograms per ml, EGF at between 715picograms per ml and 1329 picograms per ml, Eotaxin at between 37picograms per ml and 69 picograms per ml, FGF-2 at between 28 picogramsper ml and 53 picograms per ml, GM-CSF at between 7 picograms per ml and15 picograms per ml, Gro-α at between 2974 picograms per ml and 5524picograms per ml, HGF at between 352 picograms per ml and 656 picogramsper ml, IFN-α at between 253 picograms per ml and 472 picograms per ml,IFN-γ at between 33 picograms per ml and 63 picograms per ml, IL-10 atbetween 67 picograms per ml and 126 picograms per ml, IL-12p70 atbetween 9 picograms per ml and 19 picograms per ml, IL-13 at between 11picograms per ml and 22 picograms per ml, IL-15 at between 30 picogramsper ml and 57 picograms per ml, IL-17A at between 19 picograms per mland 37 picograms per ml, IL-18 at between 78 picograms per ml and 146picograms per ml, IL-1α at between 712 picograms per ml and 1324picograms per ml, IL-1β at between 14 picograms per ml and 28 picogramsper ml, IL-1RA at between 2215 picograms per ml and 4114 picograms perml, IL-2 at between 16 picograms per ml and 31 picograms per ml, IL-21at between 370 picograms per ml and 689 picograms per ml, IL-22 atbetween 16 picograms per ml and 32 picograms per ml, IL-23 at between201 picograms per ml and 376 picograms per ml, IL-27 at between 21picograms per ml and 40 picograms per ml, IL-31 at between 11 picogramsper ml and 22 picograms per ml, IL-4 at between 17 picograms per ml and33 picograms per ml, IL-5 at between 15 picograms per ml and 28picograms per ml, IL-6 at between 50 picograms per ml and 95 picogramsper ml, IL-7 at between 26 picograms per ml and 50 picograms per ml,IL-8 at between 4504 picograms per ml and 8366 picograms per ml, IL-9 atbetween 131 picograms per ml and 245 picograms per ml, IP-10 at between469 picograms per ml and 873 picograms per ml, LIF at between 41picograms per ml and 77 picograms per ml, MCP-1 at between 14,480picograms per ml and 26,893 picograms per ml, MIP-1α at between 303picograms per ml and 565 picograms per ml, MIP-1β at between 375picograms per ml and 698 picograms per ml, PDGF-BB at between 807picograms per ml and 1500 picograms per ml, PIGF-1 at between 743picograms per ml and 1381 picograms per ml, RANTES at between 3277picograms per ml and 6087 picograms per ml, SCF at between 23 picogramsper ml and 45 picograms per ml, SDF-1α at between 1114 picograms per mland 2071 picograms per ml, TNF-α at between 42 picograms per ml and 80picograms per ml, TNF-β at between 12 picograms per ml and 25 picogramsper ml, VEGF-A at between 481 picograms per ml and 895 picograms per ml,and VEGF-D at between 46 picograms per ml and 88 picograms per ml.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a process for forming a composition for implantationwithin a patient according to a first embodiment.

FIG. 2 illustrates a container having first and second layers placedtherein.

FIG. 3 illustrates a general mechanism for applying a centrifugal forceon a container.

FIG. 4 illustrates the container of FIG. 2 following the application ofa centrifugal force.

FIG. 5 illustrates the container of FIG. 4 after the removal of certainmaterial.

FIG. 6 illustrates a process for forming a composition for implantationwithin a patient according to a second embodiment.

FIG. 7 illustrates a container having first and second layers placedtherein.

FIG. 8 illustrates a general mechanism for applying a centrifugal forceon a container.

FIG. 9 illustrates the container of FIG. 7 following the application ofa centrifugal force.

FIG. 10 illustrates the container of FIG. 9 after the removal of certainmaterial.

FIG. 11 illustrates a process for forming a composition for implantationwithin a patient according to a third embodiment.

FIG. 12 illustrates first and second flexible containers during anoperation.

FIG. 13 illustrates a general mechanism for applying a centrifugal forceon the second flexible container of FIG. 12.

FIG. 14 illustrates first and second flexible containers of FIG. 12during another operation.

FIG. 15 illustrates the second flexible container following theapplication of a centrifugal force.

FIG. 16 illustrates a process for forming a composition for implantationwithin a patient according to a fourth embodiment.

FIG. 17 illustrates a container having first and second layers placedtherein.

FIG. 18 illustrates a general mechanism for applying a centrifugal forceon a container.

FIG. 19 illustrates the container of FIG. 17 following the applicationof a centrifugal force.

FIG. 20 illustrates the container of FIG. 19 after the removal ofcertain material.

FIG. 21 illustrates a process for forming a composition for implantationwithin a patient according to a fifth embodiment.

FIG. 22 illustrates a container having first and second layers placedtherein.

FIG. 23 illustrates a general mechanism for applying a centrifugal forceon a container.

FIG. 24 illustrates the container of FIG. 22 following the applicationof a centrifugal force.

FIG. 25 illustrates the container of FIG. 24 after the removal ofcertain material.

FIGS. 26A-26C illustrate two vertebrae during a spinal fusion procedurein a patient, according to an embodiment.

FIGS. 27A-27B illustrate two bone portions during a fusion procedure ina patient, according to an embodiment.

FIGS. 28A-28B illustrate soft tissue during a procedure in a patient,according to an embodiment.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

The present invention relates to a composition of cellular allograftthat is derived at least partially from umbilical cord blood (UCB), aswell as its manufacture and use. Compositions for accelerating oroptimizing biological processes or initiating new biological processesmay be produced in accordance with the teachings described herein. Cellsand growth factors may advantageously be obtained or derived fromumbilical cord blood, and may inherently benefit from the advantage thatumbilical cord blood includes native cell populations that can support auniversal donor application. An inherently increased cellular health mayalso sustain the benefit of the cellular allograft. A number ofcytokines are produced and/or maintained at controlled concentrations orprofiles within embodiments of the compositions, in order to providespecific individual and/or collaborative therapeutic functions, atparticular levels or rates.

In some embodiments of the compositions, albumin is added. It should benoted that the use of “in some embodiments” herein is intended to mean“in any of the embodiments or combinations of any of the embodiments inwhich the described element has the possibility or capability of beingadded.” In studies performed by the Applicants, albumin has been shownto increase the viability of cellular components of blood duringfreezing and thawing procedures that are common during the processing ofsuch compositions. It is thought that albumin may enhance cellularviability post-thaw by acting as a protein stabilizer, preventing theaggregation of proteins in solution as well as decreasing the amount ofincorrect protein folding. Correct folding of proteins allows theproteins to, for example, achieve their desired three-dimensionalstructure. However, misfolded proteins have modified or toxicfunctionality. Albumin has also been shown to affect the adherence ofcells to flasks used during the culturing process (culture flasks).Albumin additionally can enhance the rate of differentiation ofumbilical cord blood cells, for example stem cells. Albumin can serve asan excellent cryopreservative, at least in part because of itsantiapoptotic properties. Albumin can prevent cell death by activationof the phosphatidylinositol 3-kinase (PI3K)/AKT pathway which elicitsanti-apoptotic pro-survival pathways in different cellular systems.Aberrant ROS generation facilitates mitochondrial depolarization leadingto autophagy and apoptosis. Albumin prevents depolarization of themitochondria Albumin confers an anti-apoptotic effect through regulationof the p38 MAPK, and Akt/GSK-3β/Mcl-1/caspase-3 signaling pathways.Sustaining mTORC1 by albumin also prevents autophagic cell death. Thoughalbumin is discussed, other protectants or stabilizers may instead beused, such as dimethyl sulfoxide (DMSO) or CryoGold™. In someembodiments, a combination of protectants and stabilizers may be used.

In certain compositions a low, acidic pH, such as a pH between about 4.0and about 5.0, or about 4.4, may be desirable, such as compositionsintended for wound care that includes a cell-free extract. A low pHcomposition may have a microenvironment that promotes the death andrupture of cells, for example, during freezing or thawing. This deathand rupture of cells, or the increase thereof, may result in an increaseof soluble extracellular protein factors or bioactive agents, including,but not limited to cytokines or growth factors. The word “cytokine,”when used alone herein, can also encompass growth factors. Thesebioactive agents are thought to be a potentially helpful component inthe kinetics of wound healing. An extracellular acidic environment canstimulate Total Nucleated Cells (TNC) in umbilical cord blood, forexample, to upregulate the stress related to signaling pathwaysessential for wound healing. Cells exposed and preserved in an acidicmicroenvironment can undergo a differential transcriptional program of“regeneration” relevant genes that aid in tissue remodeling.Extracellular signal regulated kinase (ERK1/2), Mitogen activatedprotein kinase (MAPK), AKT/PKB are some of the stress induced signalingpathways that can be triggered by the acidic environment. TotalNucleated Cells in a media having the low pH, acidic environment will bein a stimulated state in response to the acidosis stress and will becapable of initiating a wound healing mechanism within a significantlyshorter time frame.

An extracellular acidic environment can also stabilize cytokines andgrowth factors present in the umbilical cord blood. Low-pH-inducedchanges can be associated with several factors, including, but notlimited to, pK(a)s, solubility limitations, eutectic crystallization,and cryoconcentration. The optimal pH of activity (the pH of maximalactivity) is correlated with the optimal pH of stability (the pH ofmaximal stability), allowing the corresponding macromolecules tosubsequently tolerate small pH fluctuations that are inevitable withcellular function. Additionally, growth factors and cytokines, such asthose disclosed herein, and including Vascular endothelial growth factor(VEGF), can bind to endothelial cells, and the extracellular matrix(ECM) increases at an acidic pH. Thus, the acidic pH in thecryopreservation will maintain the tertiary structures of theseparticular proteins in a conformation (e.g., 3-dimensional structure)that can easily bind to the cell surface rectors and initiate thesignaling cascade.

Conventional wisdom is that cells and proteins are best cryopreserved ifdone so at or very near physiological pH (neutral or near neutral), andthat shifting the pH balance to either side of the neutral pH (e.g., 7)will significantly reduce the chances of cell survival and degradeprotein activity. But these supposed degradative effects, in the low pHarea, have not been contextualized in the realm of a low grade acute andchronic stress that triggers signaling pathways. The inventors havelearned that, counterintuitively, cryopreserving at an acidic pH will“pulse” the Total Nucleated Cells to activate stress regulated signalingpathways that are strongly coupled to the physiological process of woundhealing and tissue regeneration. Thus, Total Nucleated Cells beingexposed to acidosis shock/stress (i.e., preconditioned in a lowextracellular pH) are better adapted to initiate and operate in thehealing process. Similarly, non-cellular components, particularlycytokines and growth factors which have an isoelectric point (pI) thatis in the acidic range are more stable and have a tertiary confirmationthat can easily bind to the receptors with a lower rate of dissociation(K_(D)). The low pH cryopreservatives are able to perform the two-sidedfunction of stimulating the Total Nucleated Cells and preserving lowmolecular weight proteins and growth factors, so that they can performbetter during their use (e.g., allogenic use).

The kinetics or temporal aspects of wound healing may vary depending onthe particular makeup of a composition that is added to tissue of apatient. For example, a composition containing cells that include up toabout 70% viable cells may include a larger comparative volume or amountof soluble extracellular factors/bioactive agents than a compositioncontaining cells that include greater than about 95% viable cells. Thecomposition with up to about 70% viable cells may thus cause a morerapid wound healing response than the composition with greater thanabout 95% viable cells. The use of a relatively low-pH composition asdescribed may thus be utilized in a patient with one or more chronicwounds.

On the other hand, in other compositions, a more alkaline pH, such as apH between about 7.0 and about 8.0, or about 7.5, may be desirable, suchas compositions intended to mediate a biological healing process usingviable cells, such as stem cells. Albumin is capable of helping tocontrol the effect of pH on cells, and can thus be incorporated intocompositions such as these for this purpose.

The Applicants have also observed that umbilical cord blood cells, whencultured in the presence of albumin, differentiate into severaldifferent cell types having varied morphologies. By identifying theseparticular species of cells, post-differentiation, each particular groupof cells may be enhanced to create specialty compositions in each case.For example, one or more particular compositions specially formulatedfor bone healing or bone growth, one or more particular compositionsspecially formulated for nerve healing or nerve growth, or othercompositions for healing or growth of other types of tissue. After theidentification of a cell type, the differentiation of stem cells can bedirected toward that particular cell type. With each cell type, certainbioactive agents (cytokines, growth factors, etc.) associated with thatcell type may be isolated. These bioactive agents may then be used inparticular compositions for specialized purposes. As an example,oncostatin M is a cytokine that sticks to, or binds to extracellularmatrix (ECM), and has been identified as a growth factor that affectsboth bones and nerves. For at least this reason, oncostatin M can beincorporated into bone and/or nerve allograft compositions. As anotherexample, bone morphogenetic proteins (BMP) are growth factors orcytokines that can be incorporated into allograft compositions for usewith bone.

A first embodiment of a composition comprises a solution having a firstvolume and comprising a low pH fluid base comprising dextrose. The word“base,” as used in this particular phrase, is intended to mean afoundational, basal, or holding solution, whether possessingsolvent-like properties or not. In some embodiments, a composition maycomprise a solution having a volume and comprising a low pH fluid basecomprising dextrose and dextran, and also comprising albumin, In someembodiments, the low pH fluid base may comprise LMD (Dextran 40) or anequivalent compound. In some embodiments, the low pH fluid base consistsof LIVID (Dextran 40). The low pH fluid base of dextrose and dextran,and especially the dextrose component, can act as a proteinaceousprotectant by preventing the aggregation of proteins, such as, forexample, cytokines. As described, in some embodiments, albumin may beutilized within the solution. In some embodiments utilizing albumin, thealbumin in the solution has a concentration of between about 10 mg/ml(milligram per milliliter) and about 150 mg/ml, or between about 10mg/ml (milligram per milliliter) and about 120 mg/ml. In someembodiments, the albumin in the solution has a concentration of betweenabout 15 mg/ml (milligram per milliliter) and about 30 mg/ml. In someembodiments, the albumin in the solution has a concentration of betweenabout 18 mg/ml (milligram per milliliter) and about 25 mg/ml. In someembodiments, the albumin in the solution has a concentration of about 20mg/ml. In some embodiments, the albumin in the solution has aconcentration of between about 80 mg/ml and about 150 mg/ml, or betweenabout 50 mg/ml and about 150 mg/ml. In some embodiments, the albumin inthe solution has a concentration of between about 80 mg/ml and about 120mg/ml, or between about 50 mg/ml and about 120 mg/ml, or between about90 mg/ml and about 110 mg/ml. In some embodiments, the albumin in thesolution has a concentration of between about 105 mg/ml and about 150mg/ml, or between about 105 mg/ml and about 120 mg/ml. Together, the lowpH fluid base of dextrose and dextran and the albumin act together as astorage agent or protectant (preservative, stabilizer) by maintainingthe integrity of proteins obtained from their source (e.g., umbilicalcord blood). Another beneficial use of the low pH fluid base of dextrose(and dextran, where used) is that it provides a relatively low pH. Thelow pH microenvironment provides a molecular milieu that promotes woundhealing by recruiting cells that activate signaling pathways associatedwith wound healing and tissue regeneration. In embodiments in whichalbumin is added in a powdered form to the low pH fluid base of dextrose(and dextran), a cost saving is provided, in comparison to, for example,combining the low pH fluid base of dextrose and dextran and albumin inserum.

A substance such as sodium bicarbonate powder may be added to thesolution as needed to control the final pH of the composition.Alternatively, a calcium bicarbonate liquid may be used. For example, insome embodiments, the pH of the composition may be between 4.1 and 7.2.In some embodiments, the pH of the composition may be between 4.1 and7.4, or in some cases even higher than 7.4. In some embodiments, the pHof the composition may be between 4.1 and 6.2. In some embodiments, thepH of the composition may be between 5.0 and 7.4. In some embodiments,the pH of the composition may be between 5.0 and 6.2. A pH within one ofthe ranges listed may be achieved in some cases without having to add anadditional base—in this case, the word “base” is intended to mean asubstance that releases or produces hydroxide ions (OH⁻) in aqueoussolutions—such as sodium bicarbonate or calcium bicarbonate, based onthe inherent characteristics of the low pH fluid base of dextrose anddextran. In other cases, the additional base may be used in order toachieve a pH within one of the range listed. In certain cases, it may benecessary to use an additional base in order to achieve a pH that iswithin one of the ranges listed.

In some embodiments, the albumin may be obtained or derived fromcommercially available human serum. In other embodiments, the albuminmay be obtained or derived from donor-derived serum. In someembodiments, the albumin may be obtained or derived from human umbilicalcord blood.

In some embodiments, the composition may include progenitor cells. Insome embodiments, the composition may comprise stem cells. In someembodiments, the stem cells consist of non-expanded cell populations,for example, not expanded by growth in a Petri dish or growthmedium/matrix. The overall makeup of umbilical cord blood cellpopulations and other factors may provide a broader benefit to apatient. The stem cells may comprise one or more types of stem cells,including, but not limited to, hematopoietic stem cells (HSCs) ormesenchymal stem cells (MSCs). In some embodiments, the stem cells mayinclude viable cells and non-viable cells. In some embodiments, thepercentage of viable cells among the stem cells may be greater than 70%.In some embodiments, the percentage of viable cells among the stem cellsmay be greater than 85%. In some embodiments, the percentage of viablecells among the stem cells may be greater than 95%.

In some embodiments, the composition may comprise live mononuclearcells, which may include, but are not limited to, stem cells orbioactive agents. In some embodiments, the composition comprises betweenabout 2.5 million live mononuclear cells per milliliter (ml) and about10.2 million live mononuclear cells per milliliter (ml). In someembodiments, the composition comprises between about 9.8 million livemononuclear cells per milliliter (ml) and about 10.2 million livemononuclear cells per milliliter (ml). In some embodiments, themononuclear cells may be derived from umbilical cord blood of a singledonor.

In some embodiments, the composition may comprise one or more bioactiveagents, including but not limited to cytokines or growth factors. Thebioactive agents may include any of the following: chemokines, includingmacrophage inflammatory protein alpha (MIP-1α), macrophage inflammatoryprotein beta (MIP-1β), and interferon gamma-induced protein 10 (IP-10),interleukins, including interleukin 1 Beta (IL-1β), interleukin 2(IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6(IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10(IL-10), interleukin 12 (IL-12), and interleukin 13 (IL-13),interferons, including interferon alpha (INFα), lymphokines, granulocytemacrophage colony stimulating factor (GM-CSF), stem cell factor (KL),and tumor necrosis factor alpha (TNF-α). The bioactive agents may alsoinclude any of the following: vascular endothelial growth factor (VEGF),nerve growth factor, platelet-derived growth factor (PDGF), fibroblastgrowth factors (FGF), including fibroblast growth factor 2 (FGF-2),epidermal growth factor (EGF), keratinocyte growth factor, transforminggrowth factor (TGF), insulin-like growth factor, des(1-3)-IGF-I (brainIGF-I), neurotrophin-3 (NT-3) and brain-derived neurotrophic factor(BDNF).

Other bioactive agents that may be incorporated into the composition mayinclude one or more of the following: transforming growth factor Beta 1(TGF-β1), transforming growth factor Alpha 1 (TGF-α1), interleukin 16(IL-16), platelet factor 4 (PF4), interferon gamma (IFNγ), hepatocytegrowth factor (HGF), insulin growth factor (IGF-1), fas ligand (Fas-L),monocyte chemoattractant protein 2 (MCP-2), monocyte chemoattractantprotein 3 (MCP-3), monocyte chemoattractant protein 4 (MCP-4),macrophage derived chemokine (MDC), platelet factor 4 (PF-4),thrombopoietin (TPO), insulin-like growth factor 1 (IGF-1), andinsulin-like growth factor binding protein-3 (IGFBP-3).

Other bioactive agents that may be incorporated into the composition mayinclude one or more of the following: interleukin 15 (IL-15),interleukin 17 (IL-17), interleukin 18 (IL-18), interleukin 21 (IL-21),interleukin 22 (IL-22), interleukin 23 (IL-23), interleukin 27 (IL-27),interleukin 31 (IL-31), eotaxin, CXC ligand 1 (GROα), interleukin 1receptor-alpha (IL-1RA), interleukin 1 alpha (IL-1α), leukemiainhibitory factor (LIF), placental growth factor (PIGF, PLGF), chemokineligand 5 (RANTES), stem cell factor (S.CF), stromal cell derived factor(SDF1α), and tumor necrosis factor (TNF β). Each Interleukin categorymay comprise one or more members within a category or family. Forexample, within the Interleukin 17 (IL-17) category, one of severalspecies may be chosen, such as IL-17A, IL-17B, IL-17C, IL-17D, IL-17E,or others. When IL-17 is generically denoted, it is intended toencompass any one or more of these different species.

In some embodiments, the composition may include albumin and one or morebioactive agents, wherein the albumin is derived from umbilical cordblood of a first donor, and the one or more bioactive agents are derivedfrom at least a second donor. The one or more bioactive agents may betaken or derived from commercially available human albumin and/orcommercially available human albumin serum. In some embodiments, thecomposition may include albumin and one or more bioactive agents,wherein the albumin and the one or more bioactive agents are derivedfrom umbilical cord blood of a single donor.

In some embodiments, the composition may be produced such that it doesnot comprise dimethyl sulfoxide (DMSO) to avoid any potential harmfuleffects that DMSO may have, such as inducing allergic reactions in asubject. However, in some embodiments, DMSO may still be used, forexample, as a cryoprotectant, as a replacement for Dextran, or incombination with Dextran. An alternative cryoprotectant orcryopreservative that may be substituted for Dextran and/or for DMSO, isCryoGold™, supplied by System Biosciences, Inc. of Palo Alto, Calif.,USA. Glycerol is another alternative cryoprotectant or cryopreservativethat may be substituted.

A method of producing compositions such as the embodiments describedherein is described according to a first embodiment. In step 10 of FIG.1, a sample comprising human umbilical cord blood and having a firstvolume V₁ is obtained. The human umbilical cord blood typically includesred blood cells, cells other than red blood cells, and non-cellularmaterial. In step 12, at least about 95% of the red blood cells areremoved from the sample and a substantial about of the non-cellularmaterial is removed from the sample. Step 12 produces a pelletsubstantially comprising cells other than red blood cells. The pellethas a second volume V₂. In some embodiments, at least about 98% of thered blood cells are removed from the sample. In some embodiments, atleast about 99% of the red blood cells are removed from the sample. Insome embodiments, at least about 95% (by volume) or the non-cellularmaterial is removed from the sample. In some embodiments, the removal ofred blood cells and/or non-cellular material may comprise combining thehuman umbilical cord blood with PrepaCyte®-CB solution, Bio-E, LLC,Bloomington, Minn., or an equivalent aggregation agent or sedimentationagent. In some embodiments, the removal of red blood cells and/ornon-cellular material may comprise placing the human umbilical cordblood in a container within a centrifuge, and operating the centrifuge.This may occur after combining the human umbilical cord blood withPrepaCyte-CB solution, or an equivalent aggregation agent orsedimentation agent. In step 14, at least a portion of the pellet isreconstituted by diluting it with lactated Ringer's solution. The atleast a portion of pellet has an initial volume V_(i), and the resultingdiluted portion has a diluted volume V_(dil). In some embodiments, aratio V_(dil)/V_(i) between the diluted volume and the initial volume isbetween about 0.95×V₁/V₂ and about 1.05×V₁/V₂. In step 16, a container30 (FIG. 2), which may comprise a tube configured for placement within acentrifuge, is filled with two layers. As shown in FIG. 2, a first layer32 comprises a reagent that is configured for isolating mononuclearcells. In some embodiments, the reagent comprises Ficoll-Paque®Centrifugation Media, GE Healthcare Bio-Sciences AB, Uppsala, Sweden, orequivalent media. In some embodiments, the reagent comprisesFicoll-Paque® PLUS Centrifugation Media. In some embodiments, thereagent comprises Ficoll-Paque® PREMIUM Centrifugation Media. In someembodiments, the reagent has a density of between about 1.0 grams/ml andabout 1.3 grams/ml. In some embodiments, the reagent has a density ofbetween about 1.07 grams/ml and about 1.09 grams/ml. In someembodiments, the reagent has a density of between about 1.073 grams/mland about 1.084 grams/ml. A second layer 34 is placed over the firstlayer 32. The second layer 34 comprises at least a portion of theresulting diluted portion from step 14. The first layer 32 and secondlayer 34 are placed in the container 30, such that a volume V₃ of thesecond layer 34 is placed over a volume V₄ of the first layer 32, suchthat volume V₄ is between about 15% and about 85% of V₃. In someembodiments, volume V₄ is between about 45% and about 55% of V₃. In someembodiments, volume V₄ is between about 75% and about 85% of V₃. In someembodiments, volume V₄ is about 50% of V₃.

In step 18, a sufficient centrifugal force component 48 is placed on thecontainer 30 (FIG. 3) and its contents (e.g., first layer 32 and secondlayer 34), with the centrifugal force component 48 directed toward aclosed container end 50, to cause layering as shown in FIG. 4 to form.FIG. 3 illustrates the basic mechanics of a centrifuge 46 which spinsthe container 30 in a circular path 44 around a center point 42. In FIG.4, a layer 36 comprises granulocytes and erythrocytes, and has migratedbelow a layer 32 a containing the reagent which is configured forisolating mononuclear cells, and which remains from the first layer 32of FIG. 2. Layer 38 substantially comprises mononuclear cells, and layer40 substantially comprises plasma and platelets. In some embodiments,step 18 may be performed using a centrifuge which places at least about350 times the acceleration due to gravity on the container 30 and itscontents. In some embodiments, step 18 may be performed using acentrifuge which places at least about 400 times the acceleration due togravity on the container 30 and its contents. In some embodiments, step18 may be performed using a centrifuge which places at least about 450times the acceleration due to gravity on the container 30 and itscontents. In some embodiments, the centrifuge may be run between about 5minutes and about 30 minutes to achieve the desired result. “Times theacceleration due to gravity” should be considered equivalent to relativecentrifugal force (RCF).

In addition to controlling the amount of centrifugal force on thecontents, the temperature on the contents, and/or the amount of timethat the centrifuge is operated, a user may also choose to control theramp up and ramp down (run profile) of the centrifuge; in other words,the time required to reach the chosen centrifugal force from a stoppedcondition, and the time required to reach a stopped condition from thenominally chosen centrifugal force. In a traditional centrifuge, thiscorresponds to the increase of the rotational speed (e.g., RPM) from astopped condition until a desired constant operation rate is reachedthat provides the desired relative centrifugal force, and the decreaseof the rotational speed (from a desired constant operation rate) to astopped condition (braking). This may also be referred to as a runprofile. The start-up acceleration portion of the profile may bedescribed as an acceleration curve, and the deceleration portion of theprofile may be described as a braking curve. In certain centrifuges, forexample, the acceleration may be controllable by a continuous adjustmentor may have particular set points (e.g., 1, 2, 3, 4, 5, etc.).Additionally, deceleration (e.g., braking) may by controllable bycontinuous adjustment or may have particular set points (e.g., 1, 2, 3,4, 5, etc.). The control of one or both of these two elements can beused to further optimize the effectiveness of the centrifugation. Thetime to ramp up to a desired centrifugal force from substantially zerocentrifugal force may be between about 30 seconds and about 15 minutes,or between about one minute and about five minutes, or between about 10seconds and 90 seconds. The time to ramp down from a desired centrifugalforce to substantially zero centrifugal force may be between about 30seconds and about 20 minutes, or between about one minute and about 10minutes.

In step 20 mononuclear cells from the mononuclear cell layer 38 areremoved from the container 30 and are combined with lactated Ringer'ssolution, to create a mononuclear cell solution. Prior to the removal ofthe mononuclear cells from the container 30, the layer 40 may be firstremoved (FIG. 5), thus making it easier to remove the layer 38. Care canbe taken so that the layer 38 is not disturbed when removing the layer40, for example, by use of a sterile pipette. In step 22, themononuclear cells are actively (though gently) mixed with the lactatedRinger's solution. The mononuclear cells may also be washed one, two, ormore times with lactated Ringer's solution. In some embodiments, themononuclear cell solution substantially comprises non-expanded cellpopulations. In some embodiments, the mononuclear cell solutioncomprises non-pooled cell populations. It should be noted that the layer40 is substantially removed prior to removing the layer 38 to minimizeany unnecessary contamination by platelets or plasma proteins. It shouldalso be noted that the layer 32 a is left substantially undisturbed whenremoving the layer 38, thus minimizing any unnecessary contamination bygranulocytes.

In step 22, a volume V₅ of either a portion of the mononuclear cellsolution or substantially all of the mononuclear cell solution is usedin order to estimate the number N_(L) of live cells in the volume V₅. Insome embodiments, the estimating is done by performing a CD34+ count. Insome embodiments, the estimating is done by performing a Total NucleatedCell (TNC) count. In some embodiments, the estimating is done at leastin part with an automated cell counter. Alternative methods may be usedfor performing the cell count, including: a hemtocytometer followingtrypan blue staining, an ATP test, Calcein AM, a clonogenic assay, anethidium homodimer assay, Evans blue, fluorescein diacetatehydrolysis/propidium iodide staining (FDA/PI staining), flow cytometry,formazan-based-assays (MTT/XTT), green fluorescent protein, lactatedehydrogenase (LDH), methyl violet, propidium iodide DNA stain (todifferentiate necrotic, apoptotic, and normal cells), resazurin, trypanblue which only crosses cell membranes of dead cells, or TUNEL assay. Atthe end of step 22 substantially all of the lactated Ringer's solutionmay be removed from the volume V₅. In step 24, based at least partiallyupon the estimated number N_(L) of live cells, a calculated, estimatedor determined volume V₆ of Low Molecular Weight Dextran in Dextrosesolution is added to the volume V₅ of the at least a portion of themononuclear cell solution. In some embodiments, the calculation,estimation or determination of the volume V₆ may be at least partiallybased on the assumption that approximately 75% of cells in the at leasta portion mononuclear cell solution will be viable following freezingand thawing. In some embodiments, the calculation, estimation ordetermination of the volume V₆ may be at least partially based on anintended live cell concentration of between about 2.5 million live cellsand about 10.2 million live cells per milliliter. In some embodiments,the cells may be stored cryogenically. In some embodiments, thecryogenic storage is below about −60° C. In some embodiments, thecryogenic storage is below about −80° C. In some embodiments, thecalculation, estimation or determination of the volume V₆ may be atleast partially based on an intended live cell concentration of betweenabout 2.5 million live cells and about 10.2 million live cells permilliliter, and may assume that this live cell concentration will occurafter adding albumin to the combined composition and cryogenicallystoring the combined composition. In some embodiments, the calculation,estimation or determination of the volume V₆ may be at least partiallybased on an intended live cell concentration of between about 9.8million live cells and about 10.2 million live cells per milliliter. Insome embodiments, the Low Molecular Weight Dextran in Dextrose maycomprise LMD (Dextran 40). In some embodiments, the volume V₆ addedcorresponds to the equationV ₆ =K×N _(L),

-   -   where K is between about 7.35×10⁻⁸ ml/cell and about 3.00×10⁻⁷        ml/cell, and    -   where N_(L) is the number of live cells in a 20 ml portion of        the mononuclear cell solution.

In some embodiments, the volume V₆ added corresponds to the equationV ₆ =K×N _(L),

-   -   where K is between about 7.35×10⁻⁸ ml/cell and about 7.50×10⁻⁸        ml/cell, and    -   where N_(L) is the number of live cells in a 20 ml portion of        the mononuclear cell solution.

In step 26, the pH of the combined composition of the mononuclear cellsolution and Low Molecular Weight Dextran in Dextrose solution may beadjusted if needed, in order to produce a pH within a desired range. Insome embodiments, the pH is adjusted by adding a sodium bicarbonate,such as sodium bicarbonate powder. In some embodiments, the pH isadjusted by adding a calcium bicarbonate, such as calcium bicarbonateliquid. In some embodiments, the pH is adjusted to or maintained atbetween 4.1 and 8.5. In some embodiments, the pH is adjusted to ormaintained at between 4.1 and 7.2. In some embodiments, the pH isadjusted to or maintained at between 4.1 and 6.2. In some embodiments,the pH is adjusted to or maintained at between 5.0 and 7.4. In someembodiments, the pH is adjusted to or maintained at between 5.0 and 7.2.In some embodiments, the pH is adjusted to or maintained at between 5.0and 6.2. In step 28, albumin is added to the combined composition. Insome embodiments, between about 10 mg and about 150 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 15 mg and about 30 mg of albumin may be addedper one milliliter of the combined composition. In some embodiments,between about 18 mg and about 25 mg of albumin may be added per onemilliliter of the combined composition. In some embodiments, about 20 mgof albumin may be added per one milliliter of the combined composition.

In some embodiments, the mononuclear cell solution comprises stem cells.In some embodiments, the stem cells comprise hemapoietic stem cells(HSC). In some embodiments, the stem cells comprise mesenchymal stemcells (MSC).

In some embodiments, one or more bioactive agents may be added to thecomposition. The one or more bioactive agents may include cytokines orgrowth factors, and may be derived from human umbilical cord blood. Insome embodiments, the one or more bioactive agents may be obtained orderived from the same sample comprising human umbilical cord blood thatis described in step 10. In some embodiments, the one or more bioactiveagents may be obtained or derived from human umbilical cord blood thatis not the sample comprising human umbilical cord blood that isdescribed in step 10. In some embodiments, the one or more bioactiveagents may be obtained or derived from human umbilical cord blood thatis from a different donor than the sample comprising human umbilicalcord blood that is described in step 10. In some embodiments, the one ormore bioactive agents are obtained or derived from commerciallyavailable human albumin or commercially available human albumin serum.

In some embodiments, the one or more bioactive agents may include butare not limited to cytokines or growth factors. The bioactive agents mayinclude any of the following: chemokines, including macrophageinflammatory protein alpha (MIP-1α), macrophage inflammatory proteinbeta (MIP-1β), and interferon gamma-induced protein 10 (IP-10),interleukins, including interleukin 1 Beta (IL-1β), interleukin 2(IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6(IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10(IL-10), interleukin 12 (IL-12), and interleukin 13 (IL-13),interferons, including interferon alpha (INFα), lymphokines, granulocytemacrophage colony stimulating factor (GM-CSF), stem cell factor (KL),and tumor necrosis factor alpha (TNF-α). The bioactive agents may alsoinclude any of the following: vascular endothelial growth factor (VEGF),nerve growth factor, platelet-derived growth factor (PDGF), fibroblastgrowth factors (FGF), including fibroblast growth factor 2 (FGF-2),epidermal growth factor (EGF), keratinocyte growth factor, transforminggrowth factor (TGF), insulin-like growth factor, des(1-3)-IGF-I (brainIGF-I), neurotrophin-3 (NT-3) and brain-derived neurotrophic factor(BDNF).

In some embodiments, dimethyl sulfoxide (DMSO) is not added into thecomposition in any of the steps, thus leaving the compositionsubstantially DMSO-free, and to avoid any potential harmful effects thatDMSO may have. The combined composition may then be cryogenicallystored, for example, in cryogenic vials in an ultra-low freezer. Thecryogenic storage may be controlled at a temperature below −60° C., orbelow −80° C.

A method of producing compositions such as the embodiments describedherein is described according to a second embodiment. In step 100 ofFIG. 6, a sample comprising human umbilical cord blood and having afirst volume V_(UC) is obtained. The human umbilical cord bloodtypically includes red blood cells, cells other than red blood cells,and non-cellular material. In step 102, the sample comprising humanumbilical cord blood is combined with a volume V_(PC) of PrepaCyte®-CBsolution, Bio-E, LLC, Bloomington, Minn., or an equivalent aggregationagent or sedimentation agent. Either or both of the volumes V_(UC) andV_(PC) may be selected such that the ratio V_(UC)/V_(PC) is betweenabout 0.60 and about 1.75. In some embodiments, the ratio V_(UC)/V_(PC)is between about 0.75 and about 1.25. In some embodiments, the ratioV_(UC)/V_(PC) is between about 0.60 and about 1.10. In some embodiments,the ratio V_(UC)/V_(PC) is between about 1.25 and about 1.75. Thecombined volumes V_(UC) and V_(PC) are allowed to stand undisturbed forat least about 20 minutes, or at least about 30 minutes, until asupernatant is formed, or until at least two distinct layers are formed.In step 104, a container 168 configured to be subjected to a centrifugalforce (e.g., a centrifuge tube) is partially filled with a reagent 170(FIG. 7) which is configured for isolating mononuclear cells, thereagent 170 having a volume V_(R). In some embodiments, the reagent 170comprises Ficoll-Paque® Centrifugation Media, GE Healthcare Bio-SciencesAB, Uppsala, Sweden, or equivalent media. In some embodiments, thereagent 170 comprises Ficoll-Paque® PLUS Centrifugation Media. In someembodiments, the reagent 170 comprises Ficoll-Paque® PREMIUMCentrifugation Media. In some embodiments, the reagent 170 has a densityof between about 1.0 grams/ml and about 1.3 grams/ml. In someembodiments, the reagent 170 has a density of between about 1.07grams/ml and about 1.09 grams/ml. In some embodiments, the reagent 170has a density of between about 1.073 grams/ml and about 1.084 grams/ml.The volume V_(R) of the reagent 170 represents a first layer 172 (e.g.,bottom layer) in the container 168. Further, as part of step 104, asecond layer 174 comprising a volume V_(S) of the supernatant 176produced in step 102 is layered over first layer 172 in the container168, as shown in FIG. 7. The volume V_(R) of the layer 172 of thereagent 170 is at least 67% of the volume V_(S) of the layer 174 of thesupernatant 176. In some embodiments, the volume V_(R) of the layer 172of the reagent 170 is at least 75% of the volume V_(S) of the layer 174of the supernatant 176. In some embodiments, the volume V_(R) of thelayer 172 of the reagent 170 is at least 85% of the volume V_(S) of thelayer 174 of the supernatant 176. In some embodiments, the volume V_(R)of the layer 172 of the reagent 170 is between about 30% and about 135%of the volume V_(S) of the layer 174 of the supernatant 176. In someembodiments, the volume V_(R) of the layer 172 of the reagent 170 isbetween about 50% and about 100% of the volume V_(S) of the layer 174 ofthe supernatant 176. In some embodiments, the volume V_(R) of the layer172 of the reagent 170 is between about 75% and about 100% of the volumeV_(S) of the layer 174 of the supernatant 176. The second layer 174should be added over the first layer 172 in a slow or at least gentle ornon-disruptive manner (e.g., slow speed pipetting) if a distinctinterface between the two layers 172, 174 is initially desired.

In step 106, a sufficient centrifugal force component 178 is placed onthe container 168 (FIG. 8) and its contents (e.g., first layer 172 andsecond layer 174), with the centrifugal force component 178 directedtoward a closed container end 180, to cause layering as shown in FIG. 9to form. FIG. 8 illustrates the basic mechanics of a centrifuge 46 whichspins the container 168 in a circular path 182 around a center point184. The container 168 may, for example, be carefully placed within thecentrifuge 46 in an appropriate location for spinning. In FIG. 9, alayer 186 comprises granulocytes and erythrocytes, and has migratedbelow a layer 172 a containing the reagent 170, and which remains fromthe first layer 172 of FIG. 7. Layer 188 substantially comprisesmononuclear cells, and layer 190 substantially comprises plasma andplatelets. In some embodiments, step 106 may be performed using acentrifuge which places at least about 350 times the acceleration due togravity on the container 168 and its contents. In some embodiments, step106 may be performed using a centrifuge which places at least about 400times the acceleration due to gravity on the container 168 and itscontents. In some embodiments, step 106 may be performed using acentrifuge which places at least about 450 times the acceleration due togravity on the container 168 and its contents. In some embodiments, step106 may be performed using a centrifuge which places at least about 600times the acceleration due to gravity on the container 168 and itscontents. In some embodiments, step 106 may be performed using acentrifuge which places at least about 800 times the acceleration due togravity on the container 168 and its contents. In some embodiments, step106 may be performed using a centrifuge which places at least about1,400 times the acceleration due to gravity on the container 168 and itscontents. In some embodiments, the centrifuge may be run between about 5minutes and about 30 minutes to achieve the desired result.

In addition to controlling the amount of centrifugal force on thecontents, the temperature on the contents, and/or the amount of timethat the centrifuge is operated, a user may also choose to control theramp up and ramp down (run profile) of the centrifuge; in other words,the time required to reach the chosen centrifugal force from a stoppedcondition, and the time required to reach a stopped condition from thenominally chosen centrifugal force. In a traditional centrifuge, thiscorresponds to the increase of the rotational speed (e.g., RPM) from astopped condition until a desired constant operation rate is reachedthat provides the desired relative centrifugal force, and the decreaseof the rotational speed (from a desired constant operation rate) to astopped condition (braking). This may also be referred to as a runprofile. The start-up acceleration portion of the profile may bedescribed as an acceleration curve, and the deceleration portion of theprofile may be described as a braking curve. In certain centrifuges, forexample, the acceleration may be controllable by a continuous adjustmentor may have particular set points (e.g., 1, 2, 3, 4, 5, etc.).Additionally, deceleration (e.g., braking) may by controllable bycontinuous adjustment or may have particular set points (e.g., 1, 2, 3,4, 5, etc.). The control of one or both of these two elements can beused to further optimize the effectiveness of the centrifugation. Thetime to ramp up to a desired centrifugal force from substantially zerocentrifugal force may be between about 30 seconds and about 15 minutes,or between about one minute and about five minutes, or between about 10seconds and 90 seconds. The time to ramp down from a desired centrifugalforce to substantially zero centrifugal force may be between about 30seconds and about 20 minutes, or between about one minute and about 10minutes.

In step 108, mononuclear cells from the mononuclear cell layer 188 areremoved from the container 168 and are combined with lactated Ringer'ssolution, to create a mononuclear cell solution. Prior to the removal ofthe mononuclear cells from the container 168, the layer 190 may be firstremoved (FIG. 10), thus making it easier to remove the layer 188. Carecan be taken so that the layer 188 is not disturbed when removing thelayer 190, for example, by use of a sterile pipette. In step 108, themononuclear cells from the mononuclear cell layer 188 are actively(though gently) mixed with the lactated Ringer's solution. Themononuclear cells may also be washed one, two, or more times withlactated Ringer's solution. In some embodiments, the mononuclear cellsolution substantially comprises non-expanded cell populations. In someembodiments, the mononuclear cell solution comprises non-pooled cellpopulations. It should be noted that the layer 190 is substantiallyremoved prior to removing the mononuclear cells from the layer 188 tominimize any unnecessary contamination by platelets or plasma proteins.It should also be noted that the layer 172 a is left substantiallyundisturbed when removing the layer 188, thus minimizing any unnecessarycontamination by the granulocytes in layer 186.

In step 110, a volume V_(M) of either a portion of the mononuclear cellsolution or substantially all of the mononuclear cell solution is usedin order to estimate the number N_(L) of live cells in the volume V_(M).In some embodiments, the estimating is done by performing a CD34+ count.In some embodiments, the estimating is done by performing a TotalNucleated Cell (TNC) count. In some embodiments, the estimating is doneat least in part with an automated cell counter. Alternative methods maybe used for performing the cell count, including: a hemtocytometerfollowing trypan blue staining, an ATP test, Calcein AM, a clonogenicassay, an ethidium homodimer assay, Evans blue, fluorescein diacetatehydrolysis/propidium iodide staining (FDA/PI staining), flow cytometry,formazan-based-assays (MTT/XTT), green fluorescent protein, lactatedehydrogenase (LDH), methyl violet, propidium iodide DNA stain (todifferentiate necrotic, apoptotic, and normal cells), resazurin, trypanblue which only crosses cell membranes of dead cells, or TUNEL assay. Atthe end of step 110 substantially all of the lactated Ringer's solutionmay be removed from the volume V_(M).

In step 112, albumin is added to the combined composition. In someembodiments, between about 10 mg and about 150 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 10 mg and about 120 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 50 mg and about 150 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 50 mg and about 120 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 80 mg and about 150 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 80 mg and about 120 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 105 mg and about 150 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 105 mg and about 120 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 15 mg and about 30 mg of albumin may be addedper one milliliter of the combined composition. In some embodiments,between about 18 mg and about 25 mg of albumin may be added per onemilliliter of the combined composition. In some embodiments, about 20 mgof albumin may be added per one milliliter of the combined composition.

In some embodiments, Low Molecular Weight Dextran in Dextrose solutionis added to the volume V_(M) of the at least a portion of themononuclear cell solution during or prior to the addition of albumin. Insome embodiments, the amount of Low Molecular Weight Dextran in Dextrosesolution is based at least partially upon the estimated number N_(L) oflive cells, a calculated, estimated or determined. In some embodiments,the calculation, estimation or determination of the volume V_(DD) of theLow Molecular Weight Dextran in Dextrose solution added may be at leastpartially based on the assumption that approximately 75% of cells in theat least a portion mononuclear cell solution will be viable followingfreezing and thawing. In some embodiments, the calculation, estimationor determination of the volume V_(DD) may be at least partially based onan intended live cell concentration of between about 100,000 live cellsand about 75 million live cells per milliliter. In some embodiments, thecalculation, estimation or determination of the volume V_(DD) may be atleast partially based on an intended live cell concentration of betweenabout 750,000 live cells and about 30 million live cells per milliliter.In some embodiments, the cells may be stored cryogenically. In someembodiments, the calculation, estimation or determination of the volumeV_(DD) may be at least partially based on an intended live cellconcentration of between about 10 million live cells and about 30million live cells per milliliter. In some embodiments, the calculation,estimation or determination of the volume V_(DD) may be at leastpartially based on an intended live cell concentration of between about10 million live cells and about 20 million live cells per milliliter. Insome embodiments, the cryogenic storage is below about −60° C. In someembodiments, the cryogenic storage is below about −80° C.

In some embodiments, the pH of the combined composition may be adjustedif needed, in order to produce a pH within a desired range. In someembodiments, the pH is adjusted by adding a sodium bicarbonate, such assodium bicarbonate powder. In some embodiments, the pH is adjusted byadding a calcium bicarbonate, such as calcium bicarbonate solution. Insome embodiments, the pH is adjusted to or maintained at between 4.1 and8.5. In some embodiments, the pH is adjusted to or maintained at between4.1 and 7.2. In some embodiments, the pH is adjusted to or maintained atbetween 4.1 and 6.2. In some embodiments, the pH is adjusted to ormaintained at between 5.0 and 7.4. In some embodiments, the pH isadjusted to or maintained at between 5.0 and 7.2. In some embodiments,the pH is adjusted to or maintained at between 5.0 and 6.2.

In some embodiments, the mononuclear cell solution comprises stem cells.In some embodiments, the stem cells comprise hemapoietic stem cells(HSC). In some embodiments, the stem cells comprise mesenchymal stemcells (MSC).

In some embodiments, one or more bioactive agents may be added to thecomposition. The one or more bioactive agents may include cytokines orgrowth factors, and may be derived from human umbilical cord blood. Insome embodiments, the one or more bioactive agents may be obtained orderived from the same sample comprising human umbilical cord blood thatis described in step 100. In some embodiments, the one or more bioactiveagents may be obtained or derived from human umbilical cord blood thatis not the sample comprising human umbilical cord blood that isdescribed in step 100. In some embodiments, the one or more bioactiveagents may be obtained or derived from human umbilical cord blood thatis from a different donor than the sample comprising human umbilicalcord blood that is described in step 100. In some embodiments, the oneor more bioactive agents are obtained or derived from commerciallyavailable human albumin or commercially available human albumin serum.

In some embodiments, the one or more bioactive agents may include butare not limited to cytokines or growth factors. The bioactive agents mayinclude any of the following: chemokines, including macrophageinflammatory protein alpha (MIP-1α), macrophage inflammatory proteinbeta (MIP-1β), interferon gamma-induced protein 10 (IP-10),interleukins, including interleukin 1 Beta (IL-1β), interleukin 2(IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6(IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9(IL-9), interleukin 10 (IL-10), interleukin 12 (IL-12), interleukin 13(IL-13), interleukin 15 (IL-15), interleukin 17 (IL-17), interleukin 18(IL-18), interleukin 21 (IL-21), interleukin 22 (IL-22), interleukin 23(IL-23), interleukin 27 (IL-27), interleukin 31 (IL-31), interferons,including interferon alpha (INFα), a lymphokine, granulocyte macrophagecolony stimulating factor (GM-CSF), stem cell factor (KL), tumornecrosis factor alpha (TNF-α), vascular endothelial growth factor(VEGF), nerve growth factor, platelet-derived growth factor (PDGF),fibroblast growth factor 2 (FGF-2), epidermal growth factor (EGF),keratinocyte growth factor, transforming growth factor (TGF),insulin-like growth factor, des(1-3)-IGF-I (brain IGF-I), neurotrophin-3(NT-3), brain-derived neurotrophic factor (BDNF), eotaxin, CXC ligand 1(GROα), interleukin 1 receptor-alpha (IL-1RA), interleukin 1 alpha(IL-1α), leukemia inhibitory factor (LIF), placental growth factor(PIGF, PLGF), chemokine ligand 5 (RANTES), stem cell factor (SCF),stromal cell derived factor (SDF1α), and tumor necrosis factor (TNF β).

In some embodiments, dimethyl sulfoxide (DMSO) is not added into thecomposition in any of the steps, thus leaving the compositionsubstantially DMSO-free, and to avoid any potential harmful effects thatDMSO may have. The combined composition may then be cryogenicallystored, for example, in cryogenic vials in an ultra-low freezer. Thecryogenic storage may be controlled at a temperature below −60° C., orbelow −80° C.

A method of producing compositions such as the embodiments describedherein is described according to a third embodiment. In step 200 of FIG.11, a sample comprising human umbilical cord blood and having a firstvolume V_(UC) is obtained. The human umbilical cord blood typicallyincludes red blood cells, cells other than red blood cells, andnon-cellular material. In step 202, the sample comprising humanumbilical cord blood 271 is mixed with a volume V_(PC) of PrepaCyte®-CBsolution 273, Bio-E, LLC, Bloomington, Minn., or an equivalentaggregation agent or sedimentation agent, within a first flexiblecontainer 267, as shown in FIG. 12. A spike 277 and insertion tube 279connected to the first flexible container 267 may be utilized to insertthe sample comprising human umbilical cord blood 271, or even the volumeV_(PC) of PrepaCyte®-CB solution 273. In some embodiments, the volumeV_(PC) of PrepaCyte®-CB solution 273 is received already within thefirst flexible container 267. Either or both of the volumes V_(UC) andV_(PC) may be selected such that the ratio V_(UC)/V_(PC) is betweenabout 0.60 and about 1.75. In some embodiments, the ratio V_(UC)/V_(PC)is between about 0.75 and about 1.25. In some embodiments, the ratioV_(UC)/V_(PC) is between about 0.60 and about 1.10. In some embodiments,the ratio V_(UC)/V_(PC) is between about 1.25 and about 1.75. In step204, the mixed volumes V_(UC)+V_(PC) are allowed to remain in the firstflexible container for at least 20 minutes, until a supernatant 275 isformed. In some embodiments, the mixed volumes V_(UC)+V_(PC) are allowedto remain in the first flexible container 267 for at least 30 minutes.In step 206, at least a portion of the supernatant 275 is forced fromthe from the first flexible container 267 into a second flexiblecontainer 268 (arrow) via a conduit 269 which links the first flexiblecontainer 267 to the second flexible container 268, as shown in FIG. 14.In step 208, the second flexible container 268 is placed in a centrifugeand the centrifuge is operated to apply a centrifugal force on thesupernatant within the second flexible container 268. In someembodiments, the first flexible container 267 may be removed from thesecond flexible container 268 and discarded, for example, by cutting theconduit 269. The portion of the cut conduit 269 extending from thesecond flexible container 268 may be clamped off after cutting. Asufficient centrifugal force component 278 is placed on the secondflexible container 268 (FIG. 13) and its contents, with the centrifugalforce component 278 directed toward a closed container end 280, to causelayering as shown in FIG. 15 to form. FIG. 13 illustrates the basicmechanics of a centrifuge 46 which spins the second flexible container268 in a circular path 282 around a center point 284. The secondflexible container 268 may, for example, be carefully placed within thecentrifuge 46 in an appropriate location for spinning. In FIG. 15, alayer 286 comprises granulocytes and erythrocytes and a layer 272 acontains the remaining PrepaCyte®-CB solution 273. Layer 288substantially comprises mononuclear cells, and layer 290 substantiallycomprises plasma and platelets. In some embodiments, step 208 may beperformed using a centrifuge which places at least about 350 times theacceleration due to gravity on the second flexible container 268 and itscontents. In some embodiments, step 208 may be performed using acentrifuge which places at least about 400 times the acceleration due togravity on the second flexible container 268 and its contents. In someembodiments, step 208 may be performed using a centrifuge which placesat least about 450 times the acceleration due to gravity on the secondflexible container 268 and its contents. In some embodiments, step 208may be performed using a centrifuge which places at least about 600times the acceleration due to gravity on the second flexible container268 and its contents. In some embodiments, step 208 may be performedusing a centrifuge which places at least about 800 times theacceleration due to gravity on the second flexible container 268 and itscontents. In some embodiments, step 208 may be performed using acentrifuge which places at least about 1,400 times the acceleration dueto gravity on the container 268 and its contents. In some embodiments,the centrifuge may be run between about 5 minutes and about 30 minutesto achieve the desired result.

In addition to controlling the amount of centrifugal force on thecontents, the temperature on the contents, and/or the amount of timethat the centrifuge is operated, a user may also choose to control theramp up and ramp down (run profile) of the centrifuge; in other words,the time required to reach the chosen centrifugal force from a stoppedcondition, and the time required to reach a stopped condition from thenominally chosen centrifugal force. In a traditional centrifuge, thiscorresponds to the increase of the rotational speed (e.g., RPM) from astopped condition until a desired constant operation rate is reachedthat provides the desired relative centrifugal force, and the decreaseof the rotational speed (from a desired constant operation rate) to astopped condition (braking). This may also be referred to as a runprofile. The start-up acceleration portion of the profile may bedescribed as an acceleration curve, and the deceleration portion of theprofile may be described as a braking curve. In certain centrifuges, forexample, the acceleration may be controllable by a continuous adjustmentor may have particular set points (e.g., 1, 2, 3, 4, 5, etc.).Additionally, deceleration (e.g., braking) may by controllable bycontinuous adjustment or may have particular set points (e.g., 1, 2, 3,4, 5, etc.). The control of one or both of these two elements can beused to further optimize the effectiveness of the centrifugation. Thetime to ramp up to a desired centrifugal force from substantially zerocentrifugal force may be between about 30 seconds and about 15 minutes,or between about one minute and about five minutes, or between about 10seconds and 90 seconds. The time to ramp down from a desired centrifugalforce to substantially zero centrifugal force may be between about 30seconds and about 20 minutes, or between about one minute and about 10minutes.

In step 210, mononuclear cells from the mononuclear cell layer 288 areremoved from the second flexible container 268 and are combined with areagent to create a mononuclear cell solution. In some embodiments, thereagent comprises human serum, for example, 5% human serum albumin.Prior to the removal of the mononuclear cells from the second flexiblecontainer 268, the layer 290 may be first removed, thus making it easierto remove the layer 288. Care can be taken so that the layer 288 is notdisturbed when removing the layer 290, for example, by use of a sterilepipette. The layer 290 may also be carefully removed (e.g. by aspirationwith a needle) through an additional access tube 281 having a port orvalve 283. In some embodiments, the mononuclear cell solutionsubstantially comprises non-expanded cell populations. In someembodiments, the mononuclear cell solution comprises non-pooled cellpopulations. It should be noted that the layer 290 is substantiallyremoved prior to removing the mononuclear cells from the layer 288 tominimize any unnecessary contamination by platelets or plasma proteins.It should also be noted that the layer 272 a is left substantiallyundisturbed when removing the layer 288, thus minimizing any unnecessarycontamination by the granulocytes in layer 286.

In step 212, a volume V_(M) of either a portion of the mononuclear cellsolution or substantially all of the mononuclear cell solution is usedin order to estimate the number N_(L) of live cells in the volume V_(M).In some embodiments, the estimating is done by performing a CD34+ count.In some embodiments, the estimating is done by performing a TotalNucleated Cell (TNC) count. In some embodiments, the estimating is doneat least in part with an automated cell counter. Alternative methods maybe used for performing the cell count, including: a hemtocytometerfollowing trypan blue staining, an ATP test, Calcein AM, a clonogenicassay, an ethidium homodimer assay, Evans blue, fluorescein diacetatehydrolysis/propidium iodide staining (FDA/PI staining), flow cytometry,formazan-based-assays (MTT/XTT), green fluorescent protein, lactatedehydrogenase (LDH), methyl violet, propidium iodide DNA stain (todifferentiate necrotic, apoptotic, and normal cells), resazurin, trypanblue which only crosses cell membranes of dead cells, or TUNEL assay. Atthe end of step 212 substantially all of the reagent may be removed fromthe volume V_(M).

In step 214, albumin is added to the combined composition. In someembodiments, between about 10 mg and about 150 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 10 mg and about 120 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 50 mg and about 150 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 50 mg and about 120 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 80 mg and about 150 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 80 mg and about 120 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 105 mg and about 150 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 105 mg and about 120 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 15 mg and about 30 mg of albumin may be addedper one milliliter of the combined composition. In some embodiments,between about 18 mg and about 25 mg of albumin may be added per onemilliliter of the combined composition. In some embodiments, about 20 mgof albumin may be added per one milliliter of the combined composition.

In some embodiments, Low Molecular Weight Dextran in Dextrose solutionis added to the volume V_(M) of the at least a portion of themononuclear cell solution during or prior to the addition of albumin. Insome embodiments, the amount of Low Molecular Weight Dextran in Dextrosesolution is based at least partially upon the estimated number N_(L) oflive cells, a calculated, estimated or determined. In some embodiments,the calculation, estimation or determination of the volume V_(DD) of theLow Molecular Weight Dextran in Dextrose solution added may be at leastpartially based on the assumption that approximately 75% of cells in theat least a portion mononuclear cell solution will be viable followingfreezing and thawing. In some embodiments, the calculation, estimationor determination of the volume V_(DD) may be at least partially based onan intended live cell concentration of between about 100,000 live cellsand about 75 million live cells per milliliter. In some embodiments, thecalculation, estimation or determination of the volume V_(DD) may be atleast partially based on an intended live cell concentration of betweenabout 750,000 live cells and about 30 million live cells per milliliter.In some embodiments, the cells may be stored cryogenically. In someembodiments, the calculation, estimation or determination of the volumeV_(DD) may be at least partially based on an intended live cellconcentration of between about 10 million live cells and about 30million live cells per milliliter. In some embodiments, the calculation,estimation or determination of the volume V_(DD) may be at leastpartially based on an intended live cell concentration of between about10 million live cells and about 20 million live cells per milliliter. Insome embodiments, the cryogenic storage is below about −60° C. In someembodiments, the cryogenic storage is below about −80° C.

In some embodiments, the pH of the combined composition may be adjustedif needed, in order to produce a pH within a desired range. In someembodiments, the pH is adjusted by adding a sodium bicarbonate, such assodium bicarbonate powder. In some embodiments, the pH is adjusted byadding a calcium bicarbonate, such as calcium bicarbonate solution. Insome embodiments, the pH is adjusted to or maintained at between 4.1 and8.5. In some embodiments, the pH is adjusted to or maintained at between4.1 and 7.2. In some embodiments, the pH is adjusted to or maintained atbetween 4.1 and 6.2. In some embodiments, the pH is adjusted to ormaintained at between 5.0 and 7.4. In some embodiments, the pH isadjusted to or maintained at between 5.0 and 7.2. In some embodiments,the pH is adjusted to or maintained at between 5.0 and 6.2.

In some embodiments, the mononuclear cell solution comprises stem cells.In some embodiments, the stem cells comprise hemapoietic stem cells(HSC). In some embodiments, the stem cells comprise mesenchymal stemcells (MSC).

In some embodiments, one or more bioactive agents may be added to thecomposition. The one or more bioactive agents may include cytokines orgrowth factors, and may be derived from human umbilical cord blood. Insome embodiments, the one or more bioactive agents may be obtained orderived from the same sample comprising human umbilical cord blood thatis described in step 200. In some embodiments, the one or more bioactiveagents may be obtained or derived from human umbilical cord blood thatis not the sample comprising human umbilical cord blood that isdescribed in step 200. In some embodiments, the one or more bioactiveagents may be obtained or derived from human umbilical cord blood thatis from a different donor than the sample comprising human umbilicalcord blood that is described in step 200. In some embodiments, the oneor more bioactive agents are obtained or derived from commerciallyavailable human albumin or commercially available human albumin serum.

In some embodiments, the one or more bioactive agents may include butare not limited to cytokines or growth factors. The bioactive agents mayinclude any of the following: chemokines, including macrophageinflammatory protein alpha (MIP-1α), macrophage inflammatory proteinbeta (MIP-1β), interferon gamma-induced protein 10 (IP-10),interleukins, including interleukin 1 Beta (IL-1β), interleukin 2(IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6(IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9(IL-9), interleukin 10 (IL-10), interleukin 12 (IL-12), interleukin 13(IL-13), interleukin 15 (IL-15), interleukin 17 (IL-17), interleukin 18(IL-18), interleukin 21 (IL-21), interleukin 22 (IL-22), interleukin 23(IL-23), interleukin 27 (IL-27), interleukin 31 (IL-31), interferons,including interferon alpha (INFα), a lymphokine, granulocyte macrophagecolony stimulating factor (GM-CSF), stem cell factor (KL), tumornecrosis factor alpha (TNF-α), vascular endothelial growth factor(VEGF), nerve growth factor, platelet-derived growth factor (PDGF),fibroblast growth factor 2 (FGF-2), epidermal growth factor (EGF),keratinocyte growth factor, transforming growth factor (TGF),insulin-like growth factor, des(1-3)-IGF-I (brain IGF-I), neurotrophin-3(NT-3), brain-derived neurotrophic factor (BDNF), eotaxin, CXC ligand 1(GROα), interleukin 1 receptor-alpha (IL-1RA), interleukin 1 alpha(IL-1α), leukemia inhibitory factor (LIF), placental growth factor(PIGF, PLGF), chemokine ligand 5 (RANTES), stem cell factor (SCF),stromal cell derived factor (SDF1α), and tumor necrosis factor (TNF β).

In some embodiments, dimethyl sulfoxide (DMSO) is not added into thecomposition in any of the steps, thus leaving the compositionsubstantially DMSO-free, and to avoid any potential harmful effects thatDMSO may have. The combined composition may then be cryogenicallystored, for example, in cryogenic vials in an ultra-low freezer. Thecryogenic storage may be controlled at a temperature below −60° C., orbelow −80° C.

A method of producing compositions such as the embodiments describedherein is described according to a fourth embodiment. In step 300 ofFIG. 16, a sample comprising human umbilical cord blood and having afirst volume V_(UC) is obtained. The human umbilical cord bloodtypically includes red blood cells, cells other than red blood cells,and non-cellular material. In step 302, the sample comprising humanumbilical cord blood is combined with a volume V_(PC) of PrepaCyte®-CBsolution, Bio-E, LLC, Bloomington, Minn., or an equivalent aggregationagent or sedimentation agent. Either of both of the volumes V_(UC) andV_(PC) may be selected such that the ratio V_(UC)/V_(PC) is betweenabout 0.60 and about 1.75. In some embodiments, the ratio V_(UC)/V_(PC)is between about 0.75 and about 1.25. In some embodiments, the ratioV_(UC)/V_(PC) is between about 0.60 and about 1.10. In some embodiments,the ratio V_(UC)/V_(PC) is between about 1.25 and about 1.75. Thecombined volumes V_(UC) and V_(PC) are allowed to stand undisturbed forat least about 20 minutes, or at least about 30 minutes, until asupernatant is formed, or until at least two distinct layers are formed.In step 304, a container 368 configured to be subjected to a centrifugalforce (e.g., a centrifuge tube) is partially filled with a reagent 370(FIG. 17) which is configured for isolating mononuclear cells, thereagent 370 having a volume V_(R). In some embodiments, the reagent 370comprises Ficoll-Paque® Centrifugation Media, GE Healthcare Bio-SciencesAB, Uppsala, Sweden, or equivalent media. In some embodiments, thereagent 370 comprises Ficoll-Paque® PLUS Centrifugation Media. In someembodiments, the reagent 370 comprises Ficoll-Paque® PREMIUMCentrifugation Media. In some embodiments, the reagent 370 has a densityof between about 1.0 grams/ml and about 1.3 grams/ml. In someembodiments, the reagent 370 has a density of between about 1.07grams/ml and about 1.09 grams/ml. In some embodiments, the reagent 370has a density of between about 1.073 grams/ml and about 1.084 grams/ml.The volume V_(R) of the reagent 370 represents a first layer 372 (e.g.,bottom layer) in the container 368. Further, as part of step 304, asecond layer 374 comprising a volume V_(S) of the supernatant 376produced in step 302 is layered directly over first layer 372 in thecontainer 368, as shown in FIG. 17. The layer 372 of the reagent 370 islayered with a height, or column height CH_(R), that is at least 67% ofthe height, or column height CH_(S), of the layer 374 of the supernatant376. In some embodiments, the column height CH_(R) of the layer 372 ofthe reagent 370 is at least 75% of the column height CH_(S) of the layer374 of the supernatant 376. In some embodiments, the column heightCH_(R) of the layer 372 of the reagent 370 is at least 85% of the columnheight CH_(S) of the layer 374 of the supernatant 376. In someembodiments, the column height CH_(R) of the layer 372 of the reagent370 is between about 30% and about 135% of the column height CH_(S) ofthe layer 374 of the supernatant 376. In some embodiments, the columnheight CH_(R) of the layer 372 of the reagent 370 is between about 50%and about 100% of the column height CH_(S) of the layer 374 of thesupernatant 376. In some embodiments, the column height CH_(R) of thelayer 372 of the reagent 370 is between about 75% and about 100% of thecolumn height CH_(S) of the layer 374 of the supernatant 376. The secondlayer 374 should be added over the first layer 372 in a slow or at leastgentle or non-disruptive manner (e.g., slow speed pipetting) if adistinct interface between the two layers 372, 374 is initially desired.

In step 306, a sufficient centrifugal force component 378 is placed onthe container 368 (FIG. 18) and its contents (e.g., first layer 372 andsecond layer 374), with the centrifugal force component 378 directedtoward a closed container end 380, to cause layering as shown in FIG. 19to form. FIG. 18 illustrates the basic mechanics of a centrifuge 46which spins the container 368 in a circular path 382 around a centerpoint 384. The container 368 may, for example, be carefully placedwithin the centrifuge 46 in an appropriate location for spinning. InFIG. 19, a layer 386 comprises granulocytes and erythrocytes, and hasmigrated below a layer 372 a containing the reagent 370, and whichremains from the first layer 372 of FIG. 17. Layer 388 substantiallycomprises mononuclear cells, and layer 390 substantially comprisesplasma and platelets. In some embodiments, step 306 may be performedusing a centrifuge which places at least about 350 times theacceleration due to gravity on the container 368 and its contents. Insome embodiments, step 306 may be performed using a centrifuge whichplaces at least about 400 times the acceleration due to gravity on thecontainer 368 and its contents. In some embodiments, step 306 may beperformed using a centrifuge which places at least about 450 times theacceleration due to gravity on the container 368 and its contents. Insome embodiments, step 306 may be performed using a centrifuge whichplaces at least about 600 times the acceleration due to gravity on thecontainer 368 and its contents. In some embodiments, step 306 may beperformed using a centrifuge which places at least about 800 times theacceleration due to gravity on the container 368 and its contents. Insome embodiments, step 306 may be performed using a centrifuge whichplaces at least about 1,400 times the acceleration due to gravity on thecontainer 368 and its contents. In some embodiments, the centrifuge maybe run between about 5 minutes and about 30 minutes to achieve thedesired result. In one embodiment, step 406 is performed using acentrifuge which places between about 750 times the acceleration due togravity to about 900 times the acceleration due to gravity on thecontainer 468 and its contents for a time period of between 15 minutesand 25 minutes, while maintaining the contents of the container 468 atbetween 16° C. and 22° C. In another embodiment, step 406 is performedusing a centrifuge which places between about 800 times the accelerationdue to gravity to about 850 times the acceleration due to gravity on thecontainer 468 and its contents for a time period of between 15 minutesand 25 minutes, while maintaining the contents of the container 468 atbetween 16° C. and 22° C. In another embodiment, step 406 is performedusing a centrifuge which places between about 750 times the accelerationdue to gravity to about 900 times the acceleration due to gravity on thecontainer 468 and its contents for a time period of between 15 minutesand 25 minutes, while maintaining the contents of the container 468 atbetween 17° C. and 21° C. In another embodiment, step 406 is performedusing a centrifuge which places between about 800 times the accelerationdue to gravity to about 850 times the acceleration due to gravity on thecontainer 468 and its contents for a time period of between 15 minutesand 25 minutes, while maintaining the contents of the container 468 atbetween 17° C. and 21° C.

In addition to controlling the amount of centrifugal force on thecontents, the temperature on the contents, and/or the amount of timethat the centrifuge is operated, a user may also choose to control theramp up and ramp down (run profile) of the centrifuge; in other words,the time required to reach the chosen centrifugal force from a stoppedcondition, and the time required to reach a stopped condition from thenominally chosen centrifugal force. In a traditional centrifuge, thiscorresponds to the increase of the rotational speed (e.g., RPM) from astopped condition until a desired constant operation rate is reachedthat provides the desired relative centrifugal force, and the decreaseof the rotational speed (from a desired constant operation rate) to astopped condition (braking). This may also be referred to as a runprofile. The start-up acceleration portion of the profile may bedescribed as an acceleration curve, and the deceleration portion of theprofile may be described as a braking curve. In certain centrifuges, forexample, the acceleration may be controllable by a continuous adjustmentor may have particular set points (e.g., 1, 2, 3, 4, 5, etc.).Additionally, deceleration (e.g., braking) may by controllable bycontinuous adjustment or may have particular set points (e.g., 1, 2, 3,4, 5, etc.). The control of one or both of these two elements can beused to further optimize the effectiveness of the centrifugation. Thetime to ramp up to a desired centrifugal force from substantially zerocentrifugal force may be between about 30 seconds and about 15 minutes,or between about one minute and about five minutes, or between about 10seconds and 90 seconds. The time to ramp down from a desired centrifugalforce to substantially zero centrifugal force may be between about 30seconds and about 20 minutes, or between about one minute and about 10minutes.

In step 308, mononuclear cells from the mononuclear cell layer 388 areremoved from the container 368 and are combined with lactated Ringer'ssolution, to create a mononuclear cell solution. Prior to the removal ofthe mononuclear cells from the container 368, the layer 390 may be firstremoved (FIG. 20), thus making it easier to remove the layer 388. Carecan be taken so that the layer 388 is not disturbed when removing thelayer 390, for example, by use of a sterile pipette. In step 308, themononuclear cells from the mononuclear cell layer 388 are actively(though gently) mixed with the lactated Ringer's solution. Themononuclear cells may also be washed one, two, or more times withlactated Ringer's solution. In some embodiments, the mononuclear cellsolution substantially comprises non-expanded cell populations. In someembodiments, the mononuclear cell solution comprises non-pooled cellpopulations. It should be noted that the layer 390 is substantiallyremoved prior to removing the mononuclear cells from the layer 388 tominimize any unnecessary contamination by platelets or plasma proteins.It should also be noted that the layer 372 a is left substantiallyundisturbed when removing the layer 388, thus minimizing any unnecessarycontamination by the granulocytes in layer 386.

In step 310, a volume V_(M) of either a portion of the mononuclear cellsolution or substantially all of the mononuclear cell solution is usedin order to estimate the number N_(L) of live cells in the volume V_(M).In some embodiments, the estimating is done by performing a CD34+ count.In some embodiments, the estimating is done by performing a TotalNucleated Cell (TNC) count. In some embodiments, the estimating is doneat least in part with an automated cell counter. Alternative methods maybe used for performing the cell count, including: a hemtocytometerfollowing trypan blue staining, an ATP test, Calcein AM, a clonogenicassay, an ethidium homodimer assay, Evans blue, fluorescein diacetatehydrolysis/propidium iodide staining (FDA/PI staining), flow cytometry,formazan-based-assays (MTT/XTT), green fluorescent protein, lactatedehydrogenase (LDH), methyl violet, propidium iodide DNA stain (todifferentiate necrotic, apoptotic, and normal cells), resazurin, trypanblue which only crosses cell membranes of dead cells, or TUNEL assay. Atthe end of step 310 substantially all of the lactated Ringer's solutionmay be removed from the volume V_(M).

In step 312, albumin is added to the combined composition. In someembodiments, between about 10 mg and about 150 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 10 mg and about 120 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 50 mg and about 150 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 50 mg and about 120 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 80 mg and about 150 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 80 mg and about 120 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 105 mg and about 150 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 105 mg and about 120 mg of albumin may beadded per one milliliter of the combined composition. In someembodiments, between about 15 mg and about 30 mg of albumin may be addedper one milliliter of the combined composition. In some embodiments,between about 18 mg and about 25 mg of albumin may be added per onemilliliter of the combined composition. In some embodiments, about 20 mgof albumin may be added per one milliliter of the combined composition.

In some embodiments, Low Molecular Weight Dextran in Dextrose solutionis added to the volume V_(M) of the at least a portion of themononuclear cell solution during or prior to the addition of albumin. Insome embodiments, the amount of Low Molecular Weight Dextran in Dextrosesolution is based at least partially upon the estimated number N_(L) oflive cells, a calculated, estimated or determined. In some embodiments,the calculation, estimation or determination of the volume V_(DD) of theLow Molecular Weight Dextran in Dextrose solution added may be at leastpartially based on the assumption that approximately 75% of cells in theat least a portion mononuclear cell solution will be viable followingfreezing and thawing. In some embodiments, the calculation, estimationor determination of the volume V_(DD) may be at least partially based onan intended live cell concentration of between about 100,000 live cellsand about 75 million live cells per milliliter. In some embodiments, thecalculation, estimation or determination of the volume V_(DD) may be atleast partially based on an intended live cell concentration of betweenabout 750,000 live cells and about 30 million live cells per milliliter.In some embodiments, the cells may be stored cryogenically. In someembodiments, the calculation, estimation or determination of the volumeV_(DD) may be at least partially based on an intended live cellconcentration of between about 10 million live cells and about 30million live cells per milliliter. In some embodiments, the calculation,estimation or determination of the volume V_(DD) may be at leastpartially based on an intended live cell concentration of between about10 million live cells and about 20 million live cells per milliliter. Insome embodiments, the cryogenic storage is below about −60° C. In someembodiments, the cryogenic storage is below about −80° C.

In some embodiments, the pH of the combined composition may be adjustedif needed, in order to produce a pH within a desired range. In someembodiments, the pH is adjusted by adding a sodium bicarbonate, such assodium bicarbonate powder. In some embodiments, the pH is adjusted byadding a calcium bicarbonate, such as calcium bicarbonate solution. Insome embodiments, the pH is adjusted to or maintained at between 4.1 and8.5. In some embodiments, the pH is adjusted to or maintained at between4.1 and 7.2. In some embodiments, the pH is adjusted to or maintained atbetween 4.1 and 6.2. In some embodiments, the pH is adjusted to ormaintained at between 5.0 and 7.4. In some embodiments, the pH isadjusted to or maintained at between 5.0 and 7.2. In some embodiments,the pH is adjusted to or maintained at between 5.0 and 6.2.

In some embodiments, the mononuclear cell solution comprises stem cells.In some embodiments, the stem cells comprise hemapoietic stem cells(HSC). In some embodiments, the stem cells comprise mesenchymal stemcells (MSC).

In some embodiments, one or more bioactive agents may be added to thecomposition. The one or more bioactive agents may include cytokines orgrowth factors, and may be derived from human umbilical cord blood. Insome embodiments, the one or more bioactive agents may be obtained orderived from the same sample comprising human umbilical cord blood thatis described in step 300. In some embodiments, the one or more bioactiveagents may be obtained or derived from human umbilical cord blood thatis not the sample comprising human umbilical cord blood that isdescribed in step 300. In some embodiments, the one or more bioactiveagents may be obtained or derived from human umbilical cord blood thatis from a different donor than the sample comprising human umbilicalcord blood that is described in step 300. In some embodiments, the oneor more bioactive agents are obtained or derived from commerciallyavailable human albumin or commercially available human albumin serum.

In some embodiments, the one or more bioactive agents may include butare not limited to cytokines or growth factors. The bioactive agents mayinclude any of the following: chemokines, including macrophageinflammatory protein alpha (MIP-1α), macrophage inflammatory proteinbeta (MIP-1β), interferon gamma-induced protein 10 (IP-10),interleukins, including interleukin 1 Beta (IL-1β), interleukin 2(IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6(IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9(IL-9), interleukin 10 (IL-10), interleukin 12 (IL-12), interleukin 13(IL-13), interleukin 15 (IL-15), interleukin 17 (IL-17), interleukin 18(IL-18), interleukin 21 (IL-21), interleukin 22 (IL-22), interleukin 23(IL-23), interleukin 27 (IL-27), interleukin 31 (IL-31), interferons,including interferon alpha (INFα), a lymphokine, granulocyte macrophagecolony stimulating factor (GM-CSF), stem cell factor (KL), tumornecrosis factor alpha (TNF-α), vascular endothelial growth factor(VEGF), nerve growth factor, platelet-derived growth factor (PDGF),fibroblast growth factor 2 (FGF-2), epidermal growth factor (EGF),keratinocyte growth factor, transforming growth factor (TGF),insulin-like growth factor, des(1-3)-IGF-I (brain IGF-I), neurotrophin-3(NT-3), brain-derived neurotrophic factor (BDNF), eotaxin, CXC ligand 1(GROα), interleukin 1 receptor-alpha (IL-1RA), interleukin 1 alpha(IL-1α), leukemia inhibitory factor (LIF), placental growth factor(PIGF, PLGF), chemokine ligand 5 (RANTES), stem cell factor (SCF),stromal cell derived factor (SDF1α), and tumor necrosis factor (TNF β).

In some embodiments, dimethyl sulfoxide (DMSO) is not added into thecomposition in any of the steps, thus leaving the compositionsubstantially DMSO-free, and to avoid any potential harmful effects thatDMSO may have. The combined composition may then be cryogenicallystored, for example, in cryogenic vials in an ultra-low freezer. Thecryogenic storage may be controlled at a temperature below −60° C., orbelow −80° C.

A method of producing compositions such as the embodiments describedherein is described according to a fifth embodiment. In step 400 of FIG.21, a sample comprising human umbilical cord blood and having a firstvolume V_(UC) is obtained. The human umbilical cord blood typicallyincludes red blood cells, cells other than red blood cells, andnon-cellular material. In step 402, the sample comprising humanumbilical cord blood is combined with a volume V_(PC) of PrepaCyte®-CBsolution, Bio-E, LLC, Bloomington, Minn., or an equivalent aggregationagent or sedimentation agent. Either or both of the volumes V_(UC) andV_(PC) may be selected such that the ratio V_(UC)/V_(PC) is betweenabout 0.60 and about 1.75. In some embodiments, the ratio V_(UC)/V_(PC)is between about 0.75 and about 1.25. In some embodiments, the ratioV_(UC)/V_(PC) is between about 0.60 and about 1.10. The combined volumesV_(UC) and V_(PC) are allowed to stand undisturbed for at least about 20minutes, or at least about 30 minutes, until a supernatant is formed, oruntil at least two distinct layers are formed. In step 404, a container468 configured to be subjected to a centrifugal force (e.g., acentrifuge tube) is partially filled with a reagent 470 (FIG. 22) whichis configured for isolating mononuclear cells, the reagent 470 having avolume V_(R). In some embodiments, the reagent 470 comprisesFicoll-Paque® Centrifugation Media, GE Healthcare Bio-Sciences AB,Uppsala, Sweden, or equivalent media. In some embodiments, the reagent470 comprises Ficoll-Paque® PLUS Centrifugation Media. In someembodiments, the reagent 470 comprises Ficoll-Paque® PREMIUMCentrifugation Media. In some embodiments, the reagent 470 has a densityof between about 1.0 grams/ml and about 1.3 grams/ml. In someembodiments, the reagent 470 has a density of between about 1.07grams/ml and about 1.09 grams/ml. In some embodiments, the reagent 470has a density of between about 1.073 grams/ml and about 1.084 grams/ml.The volume V_(R) of the reagent 470 represents a first layer 472 (e.g.,bottom layer) in the container 468. Further, as part of step 404, asecond layer 474 comprising a volume V_(S) of the supernatant 476produced in step 402 is layered directly over first layer 472 in thecontainer 468, as shown in FIG. 22. The layer 472 of the reagent 470 islayered with a height, or column height CH_(R), that is at least 67% ofthe height, or column height CH_(S), of the layer 474 of the supernatant476. In some embodiments, the column height CH_(R) of the layer 472 ofthe reagent 470 is at least 75% of the column height CH_(S) of the layer474 of the supernatant 476. In some embodiments, the column heightCH_(R) of the layer 472 of the reagent 470 is at least 85% of the columnheight CH_(S) of the layer 474 of the supernatant 476. In someembodiments, the column height CH_(R) of the layer 472 of the reagent470 is between about 30% and about 135% of the column height CH_(S) ofthe layer 474 of the supernatant 476. In some embodiments, the columnheight CH_(R) of the layer 472 of the reagent 470 is between about 50%and about 100% of the column height CH_(S) of the layer 474 of thesupernatant 476. In some embodiments, the column height CH_(R) of thelayer 472 of the reagent 470 is between about 75% and about 100% of thecolumn height CH_(S) of the layer 474 of the supernatant 476. The secondlayer 474 should be added over the first layer 472 in a slow or at leastgentle or non-disruptive manner (e.g., slow speed pipetting) if adistinct interface between the two layers 472, 474 is initially desired.

In step 406, a sufficient centrifugal force component 478 is placed onthe container 468 (FIG. 23) and its contents (e.g., first layer 472 andsecond layer 474), with the centrifugal force component 478 directedtoward a closed container end 480, to cause layering as shown in FIG. 24to form. FIG. 23 illustrates the basic mechanics of a centrifuge 46which spins the container 468 in a circular path 482 around a centerpoint 484. The container 468 may, for example, be carefully placedwithin the centrifuge 46 in an appropriate location for spinning. InFIG. 24, a layer 486 comprises granulocytes and erythrocytes, and hasmigrated below a layer 472 a containing the reagent 470, and whichremains from the first layer 472 of FIG. 22. Layer 488 substantiallycomprises mononuclear cells, and layer 490 substantially comprisesplasma and platelets. In some embodiments, step 406 may be performedusing a centrifuge which places at least about 350 times theacceleration due to gravity on the container 468 and its contents. Insome embodiments, step 406 may be performed using a centrifuge whichplaces at least about 400 times the acceleration due to gravity on thecontainer 468 and its contents. In some embodiments, step 406 may beperformed using a centrifuge which places at least about 450 times theacceleration due to gravity on the container 468 and its contents. Insome embodiments, step 406 may be performed using a centrifuge whichplaces at least about 600 times the acceleration due to gravity on thecontainer 468 and its contents. In some embodiments, step 406 may beperformed using a centrifuge which places at least about 800 times theacceleration due to gravity on the container 468 and its contents. Insome embodiments, step 406 may be performed using a centrifuge whichplaces at least about 1,400 times the acceleration due to gravity on thecontainer 468 and its contents. In some embodiments, the centrifuge maybe run between about 5 minutes and about 30 minutes to achieve thedesired result. In one embodiment, step 406 is performed using acentrifuge which places between about 750 times the acceleration due togravity to about 900 times the acceleration due to gravity on thecontainer 468 and its contents for a time period of between 15 minutesand 25 minutes, while maintaining the contents of the container 468 atbetween 16° C. and 22° C. In another embodiment, step 406 is performedusing a centrifuge which places between about 800 times the accelerationdue to gravity to about 850 times the acceleration due to gravity on thecontainer 468 and its contents for a time period of between 15 minutesand 25 minutes, while maintaining the contents of the container 468 atbetween 16° C. and 22° C. In another embodiment, step 406 is performedusing a centrifuge which places between about 750 times the accelerationdue to gravity to about 900 times the acceleration due to gravity on thecontainer 468 and its contents for a time period of between 15 minutesand 25 minutes, while maintaining the contents of the container 468 atbetween 17° C. and 21° C. In another embodiment, step 406 is performedusing a centrifuge which places between about 800 times the accelerationdue to gravity to about 850 times the acceleration due to gravity on thecontainer 468 and its contents for a time period of between 15 minutesand 25 minutes, while maintaining the contents of the container 468 atbetween 17° C. and 21° C.

In addition to controlling the amount of centrifugal force on thecontents, the temperature on the contents, and/or the amount of timethat the centrifuge is operated, a user may also choose to control theramp up and ramp down (run profile) of the centrifuge; in other words,the time required to reach the chosen centrifugal force from a stoppedcondition, and the time required to reach a stopped condition from thenominally chosen centrifugal force. In a traditional centrifuge, thiscorresponds to the increase of the rotational speed (e.g., RPM) from astopped condition until a desired constant operation rate is reachedthat provides the desired relative centrifugal force, and the decreaseof the rotational speed (from a desired constant operation rate) to astopped condition (braking). This may also be referred to as a runprofile. The start-up acceleration portion of the profile may bedescribed as an acceleration curve, and the deceleration portion of theprofile may be described as a braking curve. In certain centrifuges, forexample, the acceleration may be controllable by a continuous adjustmentor may have particular set points (e.g., 1, 2, 3, 4, 5, etc.).Additionally, deceleration (e.g., braking) may by controllable bycontinuous adjustment or may have particular set points (e.g., 1, 2, 3,4, 5, etc.). The control of one or both of these two elements can beused to further optimize the effectiveness of the centrifugation. Thetime to ramp up to a desired centrifugal force from substantially zerocentrifugal force may be between about 30 seconds and about 15 minutes,or between about one minute and about five minutes, or between about 10seconds and 90 seconds. The time to ramp down from a desired centrifugalforce to substantially zero centrifugal force may be between about 30seconds and about 20 minutes, or between about one minute and about 10minutes.

In step 408, mononuclear cells from the mononuclear cell layer 488 areremoved from the container 468 and are combined with lactated Ringer'ssolution, to create a mononuclear cell solution. Prior to the removal ofthe mononuclear cells from the container 468, the layer 490 may be firstremoved (FIG. 25), thus making it easier to remove the layer 488. Carecan be taken so that the layer 488 is not disturbed when removing thelayer 490, for example, by use of a sterile pipette. In step 408, themononuclear cells from the mononuclear cell layer 488 are actively(though gently) mixed with the lactated Ringer's solution. Themononuclear cells may also be washed one, two, or more times withlactated Ringer's solution. In some embodiments, the mononuclear cellsolution substantially comprises non-expanded cell populations. In someembodiments, the mononuclear cell solution comprises non-pooled cellpopulations. It should be noted that the layer 490 is substantiallyremoved prior to removing the mononuclear cells from the layer 488 tominimize any unnecessary contamination by platelets or plasma proteins.It should also be noted that the layer 472 a is left substantiallyundisturbed when removing the layer 488, thus minimizing any unnecessarycontamination by the granulocytes in layer 486.

In step 410, a volume V_(M) of either a portion of the mononuclear cellsolution or substantially all of the mononuclear cell solution is usedin order to estimate the number N_(L) of live cells in the volume V_(M).In some embodiments, the estimating is done by performing a CD34+ count.In some embodiments, the estimating is done by performing a TotalNucleated Cell (TNC) count. In some embodiments, the estimating is doneat least in part with an automated cell counter. Alternative methods maybe used for performing the cell count, including: a hemtocytometerfollowing trypan blue staining, an ATP test, Calcein AM, a clonogenicassay, an ethidium homodimer assay, Evans blue, fluorescein diacetatehydrolysis/propidium iodide staining (FDA/PI staining), flow cytometry,formazan-based-assays (MTT/XTT), green fluorescent protein, lactatedehydrogenase (LDH), methyl violet, propidium iodide DNA stain (todifferentiate necrotic, apoptotic, and normal cells), resazurin, trypanblue which only crosses cell membranes of dead cells, or TUNEL assay. Atthe end of step 410 substantially all of the lactated Ringer's solutionmay be removed from the volume V_(M).

In some embodiments, Low Molecular Weight Dextran in Dextrose solutionis added to the volume V_(M) of the at least a portion of themononuclear cell solution. In some embodiments, the amount of LowMolecular Weight Dextran in Dextrose solution is based at leastpartially upon the estimated number N_(L) of live cells, a calculated,estimated or determined. In some embodiments, the calculation,estimation or determination of the volume V_(DD) of the Low MolecularWeight Dextran in Dextrose solution added may be at least partiallybased on the assumption that approximately 75% of cells in the at leasta portion mononuclear cell solution will be viable following freezingand thawing. In some embodiments, the calculation, estimation ordetermination of the volume V_(DD) may be at least partially based on anintended live cell concentration of between about 100,000 live cells andabout 75 million live cells per milliliter. In some embodiments, thecalculation, estimation or determination of the volume V_(DD) may be atleast partially based on an intended live cell concentration of betweenabout 750,000 live cells and about 30 million live cells per milliliter.In some embodiments, the cells may be stored cryogenically. In someembodiments, the calculation, estimation or determination of the volumeV_(DD) may be at least partially based on an intended live cellconcentration of between about 10 million live cells and about 30million live cells per milliliter. In some embodiments, the calculation,estimation or determination of the volume V_(DD) may be at leastpartially based on an intended live cell concentration of between about10 million live cells and about 20 million live cells per milliliter. Insome embodiments, the cryogenic storage is below about −60° C. In someembodiments, the cryogenic storage is below about −80° C.

In some embodiments, the pH of the combined composition may be adjustedif needed, in order to produce a pH within a desired range. In someembodiments, the pH is adjusted by adding a sodium bicarbonate, such assodium bicarbonate powder. In some embodiments, the pH is adjusted byadding a calcium bicarbonate, such as calcium bicarbonate solution. Insome embodiments, the pH is adjusted to or maintained at between 4.1 and8.5. In some embodiments, the pH is adjusted to or maintained at between4.1 and 7.2. In some embodiments, the pH is adjusted to or maintained atbetween 4.1 and 6.2. In some embodiments, the pH is adjusted to ormaintained at between 5.0 and 7.4. In some embodiments, the pH isadjusted to or maintained at between 5.0 and 7.2. In some embodiments,the pH is adjusted to or maintained at between 5.0 and 6.2.

In some embodiments, the mononuclear cell solution comprises stem cells.In some embodiments, the stem cells comprise hemapoietic stem cells(HSC). In some embodiments, the stem cells comprise mesenchymal stemcells (MSC).

In some embodiments, one or more bioactive agents may be added to thecomposition. The one or more bioactive agents may include cytokines orgrowth factors, and may be derived from human umbilical cord blood. Insome embodiments, the one or more bioactive agents may be obtained orderived from the same sample comprising human umbilical cord blood thatis described in step 400. In some embodiments, the one or more bioactiveagents may be obtained or derived from human umbilical cord blood thatis not the sample comprising human umbilical cord blood that isdescribed in step 400. In some embodiments, the one or more bioactiveagents may be obtained or derived from human umbilical cord blood thatis from a different donor than the sample comprising human umbilicalcord blood that is described in step 400. In some embodiments, the oneor more bioactive agents are obtained or derived from commerciallyavailable human albumin or commercially available human albumin serum.

In some embodiments, the one or more bioactive agents may include butare not limited to cytokines or growth factors. The bioactive agents mayinclude any of the following: chemokines, including macrophageinflammatory protein alpha (MIP-1α), macrophage inflammatory proteinbeta (MIP-1β), interferon gamma-induced protein 10 (IP-10),interleukins, including interleukin 1 Beta (IL-1β), interleukin 2(IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6(IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9(IL-9), interleukin 10 (IL-10), interleukin 12 (IL-12), interleukin 13(IL-13), interleukin 15 (IL-15), interleukin 17 (IL-17), interleukin 18(IL-18), interleukin 21 (IL-21), interleukin 22 (IL-22), interleukin 23(IL-23), interleukin 27 (IL-27), interleukin 31 (IL-31), interferons,including interferon alpha (INFα), a lymphokine, granulocyte macrophagecolony stimulating factor (GM-CSF), stem cell factor (KL), tumornecrosis factor alpha (TNF-α), vascular endothelial growth factor(VEGF), nerve growth factor, platelet-derived growth factor (PDGF),fibroblast growth factor 2 (FGF-2), epidermal growth factor (EGF),keratinocyte growth factor, transforming growth factor (TGF),insulin-like growth factor, des(1-3)-IGF-I (brain IGF-I), neurotrophin-3(NT-3), brain-derived neurotrophic factor (BDNF), eotaxin, CXC ligand 1(GROα), interleukin 1 receptor-alpha (IL-1RA), interleukin 1 alpha(IL-1α), leukemia inhibitory factor (LIF), placental growth factor(PIGF, PLGF), chemokine ligand 5 (RANTES), stem cell factor (SCF),stromal cell derived factor (SDF1α), and tumor necrosis factor (TNF β).

In some embodiments, dimethyl sulfoxide (DMSO) is not added into thecomposition in any of the steps, thus leaving the compositionsubstantially DMSO-free, and to avoid any potential harmful effects thatDMSO may have. The combined composition may then be cryogenicallystored, for example, in cryogenic vials in an ultra-low freezer. Thecryogenic storage may be controlled at a temperature below −60° C., orbelow −80° C.

In some embodiments, the amount of Low Molecular Weight Dextrosesolution having a low pH is based at least partially upon the estimatednumber N_(L) of live cells, a calculated, estimated or determined,Furthermore, the sedimentation processing and centrifugation techniquesdescribed herein to optimize concentrations of one or more particularcytokines within the final composition. Centrifugation processes can beused to enrich the mononuclear cell layer, but can also be used toobtain the extracellular cytokines and growth factors associated withthe mononuclear cell layer. The initial nature and concentration of thecytokines (e.g., growth factors, etc.) present in the final collectedlayer are typically independent of the viable cell count associated withthis layer. Physiological stimuli can release a significant amount ofcytokines into circulation that will eventually be present in umbilicalcord blood. Physical conditions may be controlled during the productionof the compositions described herein to control the concentrations ofone or more particular cytokines. External factors may include ambienttemperature of collection, ambient temperature of storage, ambienttemperature of transport, and ambient temperature of processing.External factors may include the existence of particular anti-coagulants(EDTA, etc). The total time of each portion of the process may also actto control the amount of cytokines, including the total time durationbetween collection of cord blood and final processing, or the total timeduration of processing. The effects from controlling centrifugation(e.g., controlling applied centrifugal force) can be used to optimizethe formation of the mononuclear cell layer. The centrifuge speed canalso be adjusted to adjust cytokine composition/configuration orcytokine concentration. The physio-chemical properties of any diluentsused or other factors in which cells are suspended can help determinethe biological half-life of the cytokines.

For example, step 406 is performed using a centrifuge which placesbetween about 750 times the acceleration due to gravity to about 900times the acceleration due to gravity on the container 468 and itscontents for a time period of between 15 minutes and 25 minutes, whilemaintaining the contents of the container 468 at between 16° C. and 22°C. In another embodiment, step 406 is performed using a centrifuge whichplaces between about 800 times the acceleration due to gravity to about850 times the acceleration due to gravity on the container 468 and itscontents for a time period of between 15 minutes and 25 minutes, whilemaintaining the contents of the container 468 at between 16° C. and 22°C. In another embodiment, step 406 is performed using a centrifuge whichplaces between about 750 times the acceleration due to gravity to about900 times the acceleration due to gravity on the container 468 and itscontents for a time period of between 15 minutes and 25 minutes, whilemaintaining the contents of the container 468 at between 17° C. and 21°C. In another embodiment, step 406 is performed using a centrifuge whichplaces between about 800 times the acceleration due to gravity to about850 times the acceleration due to gravity on the container 468 and itscontents for a time period of between 15 minutes and 25 minutes, whilemaintaining the contents of the container 468 at between 17° C. and 21°C.

The time to ramp up to a desired centrifugal force from substantiallyzero centrifugal force may be between about 30 seconds and about 15minutes, or between about one minute and about five minutes, or betweenabout 10 seconds and 90 seconds. The time to ramp down from a desiredcentrifugal force to substantially zero centrifugal force may be betweenabout 30 seconds and about 20 minutes, or between about one minute andabout 10 minutes.

Particular steps in aliquoting mononuclear cells can be varied slightlyto vary concentrations of multiple different cytokines within thecomposition, such as controlling the concentration ranges of twodifferent cytokines, three different cytokines, four differentcytokines, five different cytokines, six different cytokines, sevendifferent cytokines, eight different cytokines, nine differentcytokines, ten different cytokines, and up to the 45 differentcytokines, listed below. Effective concentrations within the compositionof particular cytokines are listed in Table 1 below.

TABLE 1 Minimum Maximum Concen- Concen- tration tration Cytokine (pico-(pico- Symbol Cytokine grams/ml) grams/ml) BDNF Brain-derivedneurotrophic factor 492 915 bNGF Beta-nerve growth factor 14 26 EGFEpidermal growth factor 715 1329 Eotaxin Eotaxin 37 69 FGF-2 Fibroblastgrowth factor-2 28 53 GM-CSF Granulocyte-macrophage colony 7 15stimulating factor Gro-alpha Growth-regulated alpha protein 2974 5524HGF Hepatocyte growth factor 352 656 IFN-alpha Interferon alpha 253 472IFN-gamma Interferon gamma 33 62 IL-10 Interleukin-10 67 126 IL-12p70Interleukin-12, heterodimer p70 9 19 IL-13 Interleukin-13 11 22 IL-15Interleukin-15 30 57 IL-17A Interleukin-17A 19 37 IL-18 Interleukin-1878 146 IL-1alpha Interleukin-1 alpha 712 1324 IL-1beta Interleukin-1beta 14 28 IL-1RA Interleukin-1 receptor-alpha 2215 4114 IL-2Interleukin-2 16 31 IL-21 Interleukin-21 370 689 IL-22 Interleukin-22 1632 IL-23 Interleukin-23 201 376 IL-27 Interleukin-27 21 40 IL-31Interleukin-31 11 22 IL-4 Interleukin-4 17 33 IL-5 Interleukin-5 15 28IL-6 Interleukin-6 50 95 IL-7 Interleukin-7 26 50 IL-8 Interleukin-84504 8366 IL-9 Interleukin-9 131 245 IP-10 Interferon gamma-induced 469873 protein-10 LIF Leukemia inhibitory factor 41 77 MCP-1 Monocytechemoattractant 14,480 26,893 protein-1 MIP-1alpha Macrophageinflammatory 303 565 protein-1 alpha MIP-1beta Macrophage inflammatory375 698 protein-1 beta PDGF-BB Platelet-derived growth factor 807 1500subunit B-heterodimer PIGF-1 Placenta growth factor-1 743 1381 RANTESChemokine (C-C motif) ligand 5 3277 6087 SCF SCF complex 23 45SDF-1alpha Stromal cell-derived factor-1 alpha 1114 2071 TNF-alpha Tumornecrosis factor alpha 42 80 TNF-beta Tumor necrosis factor beta 12 25VEGF-A Vascular endothelial growth 481 895 factor-A VEGF-D Vascularendothelial growth 46 88 factor-D

In addition to optimization of concentrations of one or more particularcytokines within the final composition, one or more inspection processmay also be used to reject compositions produced wherein one or morecytokines within the composition is at an undesirable concentration,such as too high or too low. In some embodiments, one part of thecomposition is diluted with four parts of mesenchymal stem cell growthmedia. The dilution is then incubated for at least 24 hours, or in otherembodiments, for at least 48 hours, in a humidified chamber. Thehumidified chamber in some embodiments includes an environment ofbetween about 5% carbon di-oxide and is maintained at a temperature ofabout 37° C. The dilution (also known as conditioned media) is used in aassay to determine the concentrations of the various cytokines. In someembodiments, a Luminex® MAGPIC® system may be used for performing theassay. The normalized concentrations determined in the assay are thanmultiplied by a factor of 5, to determine the concentrations of thecytokines in the undilute composition. The concentrations may beexpressed in units of picograms per milliliter (picograms/ml). Analternative composition may be produced by varying particular steps inaliquoting mononuclear cells and/or by use of the inspection andacceptance or rejection process, to produce effective concentrationswithin the composition of particular cytokines, as listed in Table 2below.

TABLE 2 Minimum Maximum Concen- Concen- tration tration Cytokine (pico-(pico- Symbol Cytokine grams/ml) grams/ml) BDNF Brain-derivedneurotrophic factor 527 880 bNGF Beta-nerve growth factor 15 25 EGFEpidermal growth factor 766 1278 Eotaxin Eotaxin 39 67 FGF-2 Fibroblastgrowth factor-2 30 51 GM-CSF Granulocyte-macrophage colony 8 15stimulating factor Gro-alpha Growth-regulated alpha protein 3186 5312HGF Hepatocyte growth factor 378 631 IFN-alpha Interferon alpha 271 454IFN-gamma Interferon gamma 35 60 IL-10 Interleukin-10 72 121 IL-12p70Interleukin-12, heterodimer p70 10 18 IL-13 Interleukin-13 12 21 IL-15Interleukin-15 32 55 IL-17A Interleukin-17A 21 35 IL-18 Interleukin-1883 140 IL-1alpha Interleukin-1 alpha 763 1273 IL-1beta Interleukin-1beta 15 27 IL-1RA Interleukin-1 receptor-alpha 2373 3956 IL-2Interleukin-2 17 30 IL-21 Interleukin-21 396 662 IL-22 Interleukin-22 1730 IL-23 Interleukin-23 216 361 IL-27 Interleukin-27 22 38 IL-31Interleukin-31 12 21 IL-4 Interleukin-4 18 32 IL-5 Interleukin-5 16 27IL-6 Interleukin-6 54 91 IL-7 Interleukin-7 28 48 IL-8 Interleukin-84826 8044 IL-9 Interleukin-9 141 236 IP-10 Interferon gamma-induced 503840 protein-10 LIF Leukemia inhibitory factor 44 74 MCP-1 Monocytechemoattractant 15,514 25,859 protein-1 MIP-1alpha Macrophageinflammatory 325 543 protein-1 alpha MIP-1beta Macrophage inflammatory402 671 protein-1 beta PDGF-BB Platelet-derived growth factor 865 1442subunit B-heterodimer PIGF-1 Placenta growth factor-1 796 1328 RANTESChemokine (C-C motif) ligand 5 3511 5853 SCF SCF complex 25 43SDF-1alpha Stromal cell-derived factor-1 alpha 1194 1991 TNF-alpha Tumornecrosis factor alpha 45 77 TNF-beta Tumor necrosis factor beta 13 24VEGF-A Vascular endothelial growth 516 861 factor-A VEGF-D Vascularendothelial growth 50 84 factor-D

Most or all of these cytokines as quantified in the above ranges act asimportant signaling molecules that regulate biological functionsassociated with tissue regeneration, such as cell proliferation, cellmigration, chemotaxis, angiogenesis, and remodeling. Many of thecytokines listed have at least some overlapping functions. For example,both PDGF and VEGF can support angiogenesis. Some cytokines can performmore than one function. For example, PDGF can be important in bothproliferation and angiogenesis. An alternative composition may beproduced by varying particular steps in aliquoting mononuclear cellsand/or by use of the inspection and acceptance or rejection process, toproduce effective concentrations within the composition of particularcytokines, as listed in Table 3 below.

TABLE 3 Minimum Maximum Concen- Concen- tration tration Cytokine (pico-(pico- Symbol Cytokine grams/ml) grams/ml) BDNF Brain-derivedneurotrophic factor 562 844 bNGF Beta-nerve growth factor 16 24 EGFEpidermal growth factor 817 1227 Eotaxin Eotaxin 42 64 FGF-2 Fibroblastgrowth factor-2 32 49 GM-CSF Granulocyte-macrophage colony 9 14stimulating factor Gro-alpha Growth-regulated alpha protein 3399 5099HGF Hepatocyte growth factor 403 605 IFN-alpha Interferon alpha 290 436IFN-gamma Interferon gamma 38 57 IL-10 Interleukin-10 77 117 IL-12p70Interleukin-12, heterodimer p70 11 18 IL-13 Interleukin-13 13 20 IL-15Interleukin-15 34 53 IL-17A Interleukin-17A 22 34 IL-18 Interleukin-1889 135 IL-1alpha Interleukin-1 alpha 814 1222 IL-1beta Interleukin-1beta 17 26 IL-1RA Interleukin-1 receptor-alpha 2531 3798 IL-2Interleukin-2 18 29 IL-21 Interleukin-21 423 636 IL-22 Interleukin-22 1929 IL-23 Interleukin-23 230 347 IL-27 Interleukin-27 24 37 IL-31Interleukin-31 13 20 IL-4 Interleukin-4 19 30 IL-5 Interleukin-5 17 26IL-6 Interleukin-6 58 88 IL-7 Interleukin-7 30 46 IL-8 Interleukin-85147 7722 IL-9 Interleukin-9 150 226 IP-10 Interferon gamma-induced 537806 protein-10 LIF Leukemia inhibitory factor 47 71 MCP-1 Monocytechemoattractant 16,549 24,824 protein-1 MIP-1alpha Macrophageinflammatory 347 522 protein-1 alpha MIP-1beta Macrophage inflammatory429 644 protein-1 beta PDGF-BB Platelet-derived growth factor 922 1385subunit B-heterodimer PIGF-1 Placenta growth factor-1 849 1275 RANTESChemokine (C-C motif) ligand 5 3745 5619 SCF SCF complex 27 42SDF-1alpha Stromal cell-derived factor-1 alpha 1273 1911 TNF-alpha Tumornecrosis factor alpha 49 74 TNF-beta Tumor necrosis factor beta 14 23VEGF-A Vascular endothelial growth 550 826 factor-A VEGF-D Vascularendothelial growth 53 81 factor-D

An alternative composition may be produced by varying particular stepsin aliquoting mononuclear cells and/or by use of the inspection andacceptance or rejection process, to produce effective concentrationswithin the composition of particular cytokines, as listed in Table 4below.

TABLE 4 Minimum Maximum Concen- Concen- tration tration Cytokine (pico-(pico- Symbol Cytokine grams/ml) grams/ml) BDNF Brain-derivedneurotrophic factor 597 809 bNGF Beta-nerve growth factor 17 23 EGFEpidermal growth factor 868 1176 Eotaxin Eotaxin 45 61 FGF-2 Fibroblastgrowth factor-2 34 47 GM-CSF Granulocyte-macrophage colony 9 13stimulating factor Gro-alpha Growth-regulated alpha protein 3611 4887HGF Hepatocyte growth factor 428 580 IFN-alpha Interferon alpha 308 418IFN-gamma Interferon gamma 40 55 IL-10 Interleukin-10 82 112 IL-12p70Interleukin-12, heterodimer p70 12 17 IL-13 Interleukin-13 13 19 IL-15Interleukin-15 36 51 IL-17A Interleukin-17A 23 33 IL-18 Interleukin-1895 129 IL-1alpha Interleukin-1 alpha 865 1171 IL-1beta Interleukin-1beta 18 25 IL-1RA Interleukin-1 receptor-alpha 2689 3640 IL-2Interleukin-2 20 28 IL-21 Interleukin-21 449 609 IL-22 Interleukin-22 2028 IL-23 Interleukin-23 245 332 IL-27 Interleukin-27 25 35 IL-31Interleukin-31 13 19 IL-4 Interleukin-4 21 29 IL-5 Interleukin-5 18 25IL-6 Interleukin-6 61 84 IL-7 Interleukin-7 32 44 IL-8 Interleukin-85469 7401 IL-9 Interleukin-9 160 217 IP-10 Interferon gamma-induced 570772 protein-10 LIF Leukemia inhibitory factor 50 68 MCP-1 Monocytechemoattractant 17,583 23,790 protein-1 MIP-1alpha Macrophageinflammatory 369 500 protein-1 alpha MIP-1beta Macrophage inflammatory455 617 protein-1 beta PDGF-BB Platelet-derived growth factor 980 1327subunit B-heterodimer PIGF-1 Placenta growth factor-1 902 1222 RANTESChemokine (C-C motif) ligand 5 3979 5385 SCF SCF complex 29 40SDF-1alpha Stromal cell-derived factor-1 alpha 1353 1832 TNF-alpha Tumornecrosis factor alpha 52 71 TNF-beta Tumor necrosis factor beta 15 22VEGF-A Vascular endothelial growth 585 792 factor-A VEGF-D Vascularendothelial growth 56 78 factor-D

An alternative composition may be produced by varying particular stepsin aliquoting mononuclear cells and/or by use of the inspection andacceptance or rejection process, to produce effective concentrationswithin the composition of particular cytokines, as listed in Table 5below.

TABLE 5 Minimum Maximum Concen- Concen- tration tration Cytokine (pico-(pico- Symbol Cytokine grams/ml) grams/ml) BDNF Brain-derivedneurotrophic factor 632 774 bNGF Beta-nerve growth factor 18 22 EGFEpidermal growth factor 919 1124 Eotaxin Eotaxin 47 59 FGF-2 Fibroblastgrowth factor-2 36 45 GM-CSF Granulocyte-macrophage colony 10 13stimulating factor Gro-alpha Growth-regulated alpha protein 3823 4674HGF Hepatocyte growth factor 453 555 IFN-alpha Interferon alpha 326 399IFN-gamma Interferon gamma 42 53 IL-10 Interleukin-10 87 107 IL-12p70Interleukin-12, heterodimer p70 12 16 IL-13 Interleukin-13 14 19 IL-15Interleukin-15 39 48 IL-17A Interleukin-17A 25 31 IL-18 Interleukin-18100 124 IL-1alpha Interleukin-1 alpha 916 1121 IL-1beta Interleukin-1beta 19 24 IL-1RA Interleukin-1 receptor-alpha 2848 3481 IL-2Interleukin-2 21 26 IL-21 Interleukin-21 476 583 IL-22 Interleukin-22 2127 IL-23 Interleukin-23 259 318 IL-27 Interleukin-27 27 34 IL-31Interleukin-31 14 18 IL-4 Interleukin-4 22 28 IL-5 Interleukin-5 19 24IL-6 Interleukin-6 65 81 IL-7 Interleukin-7 34 42 IL-8 Interleukin-85791 7079 IL-9 Interleukin-9 169 208 IP-10 Interferon gamma-induced 604739 protein-10 LIF Leukemia inhibitory factor 53 66 MCP-1 Monocytechemoattractant 18,617 22,756 protein-1 MIP-1alpha Macrophageinflammatory 390 478 protein-1 alpha MIP-1beta Macrophage inflammatory482 590 protein-1 beta PDGF-BB Platelet-derived growth factor 1038 1269subunit B-heterodimer PIGF-1 Placenta growth factor-1 955 1169 RANTESChemokine (C-C motif) ligand 5 4213 5151 SCF SCF complex 30 38SDF-1alpha Stromal cell-derived factor-1 alpha 1433 1752 TNF-alpha Tumornecrosis factor alpha 55 68 TNF-beta Tumor necrosis factor beta 16 21VEGF-A Vascular endothelial growth 619 758 factor-A VEGF-D Vascularendothelial growth 60 74 factor-D

An alternative composition may be produced by varying particular stepsin aliquoting mononuclear cells and/or by use of the inspection andacceptance or rejection process, to produce effective concentrationswithin the composition of particular cytokines, as listed in Table 6below.

TABLE 6 Minimum Maximum Concen- Concen- tration tration Cytokine (pico-(pico- Symbol Cytokine grams/ml) grams/ml) BDNF Brain-derivedneurotrophic factor 668 739 bNGF Beta-nerve growth factor 19 21 EGFEpidermal growth factor 970 1073 Eotaxin Eotaxin 50 56 FGF-2 Fibroblastgrowth factor-2 38 43 GM-CSF Granulocyte-macrophage colony 10 12stimulating factor Gro-alpha Growth-regulated alpha protein 4036 4462HGF Hepatocyte growth factor 478 530 IFN-alpha Interferon alpha 344 381IFN-gamma Interferon gamma 45 50 IL-10 Interleukin-10 91 102 IL-12p70Interleukin-12, heterodimer p70 13 15 IL-13 Interleukin-13 15 18 IL-15Interleukin-15 41 46 IL-17A Interleukin-17A 26 30 IL-18 Interleukin-18106 118 IL-1alpha Interleukin-1 alpha 967 1070 IL-1beta Interleukin-1beta 20 23 IL-1RA Interleukin-1 receptor-alpha 3006 3323 IL-2Interleukin-2 22 25 IL-21 Interleukin-21 502 556 IL-22 Interleukin-22 2226 IL-23 Interleukin-23 274 303 IL-27 Interleukin-27 28 32 IL-31Interleukin-31 15 18 IL-4 Interleukin-4 23 27 IL-5 Interleukin-5 20 23IL-6 Interleukin-6 69 77 IL-7 Interleukin-7 36 40 IL-8 Interleukin-86113 6757 IL-9 Interleukin-9 178 198 IP-10 Interferon gamma-induced 637705 protein-10 LIF Leukemia inhibitory factor 56 63 MCP-1 Monocytechemoattractant 19,652 21,721 protein-1 MIP-1alpha Macrophageinflammatory 412 456 protein-1 alpha MIP-1beta Macrophage inflammatory509 564 protein-1 beta PDGF-BB Platelet-derived growth factor 1095 1212subunit B-heterodimer PIGF-1 Placenta growth factor-1 1009 1116 RANTESChemokine (C-C motif) ligand 5 4447 4916 SCF SCF complex 32 36SDF-1alpha Stromal cell-derived factor-1 alpha 1512 1672 TNF-alpha Tumornecrosis factor alpha 58 65 TNF-beta Tumor necrosis factor beta 17 20VEGF-A Vascular endothelial growth 653 723 factor-A VEGF-D Vascularendothelial growth 63 71 factor-D

Cytokines of particular interest include EGF (Epidermal growth factor),PDGF-BB (Platelet-derived growth factor subunit B-heterodimer), VEGF-A(Vascular endothelial growth factor-A), SCF (SCF Complex), IL-1RA(Interleukin-1 receptor-alpha), GM-CSF (Granulocyte-macrophage colonystimulating factor), IL-4 (Interleukin-4), IL-8 (Interleukin-8),SDF-1alpha (Stromal cell-derived factor-1-alpha), and RANTES (CCL5,Chemokine (C-C motif) ligand 5). The inventors have found that bycontrolling the concentrations of some or all of these ten cytokineswith tighter control than that used in the other cytokines in the tablesabove can be effective in producing compositions with improved woundhealing qualities. For example, an alternative composition may beproduced by varying particular steps in aliquoting mononuclear cellsand/or by use of the inspection and acceptance or rejection process, toproduce effective concentrations within the composition of particularcytokines, as listed in Table 7 below.

TABLE 7 Minimum Maximum Cytokine Concentration Concentration SymbolCytokine (picograms/ml) (picograms/ml) EGF Epidermal growth factor 7151329 IL-1RA Interleukin-1 receptor-alpha 2215 4114 PDGF-BBPlatelet-derived growth 807 1500 factor subunit B-hetero- dimer SCF SCFcomplex 23 45 VEGF-A Vascular endothelial growth 481 895 factor-A

In other embodiments, an alternative composition may be produced byvarying particular steps in aliquoting mononuclear cells and/or by useof the inspection and acceptance or rejection process, to produceeffective concentrations within the composition of particular cytokines,as listed in Table 8 below.

TABLE 8 Minimum Maximum Cytokine Concentration Concentration SymbolCytokine (picograms/ml) (picograms/ml) EGF Epidermal growth factor 7661278 IL-1RA Interleukin-1 receptor-alpha 2373 3956 PDGF-BBPlatelet-derived growth 865 1442 factor subunit B-hetero- dimer SCF SCFcomplex 25 43 VEGF-A Vascular endothelial growth 516 861 factor-A

In other embodiments, an alternative composition may be produced byvarying particular steps in aliquoting mononuclear cells and/or by useof the inspection and acceptance or rejection process, to produceeffective concentrations within the composition of particular cytokines,as listed in Table 9 below.

TABLE 9 Minimum Maximum Cytokine Concentration Concentration SymbolCytokine (picograms/ml) (picograms/ml) EGF Epidermal growth factor 8171227 IL-1RA Interleukin-1 receptor-alpha 2531 3798 PDGF-BBPlatelet-derived growth 922 1385 factor subunit B-hetero- dimer SCF SCFcomplex 27 42 VEGF-A Vascular endothelial growth 550 826 factor-A

In other embodiments, an alternative composition may be produced byvarying particular steps in aliquoting mononuclear cells and/or by useof the inspection and acceptance or rejection process, to produceeffective concentrations within the composition of particular cytokines,as listed in Table 10 below.

TABLE 10 Minimum Maximum Cytokine Concentration Concentration SymbolCytokine (picograms/ml) (picograms/ml) EGF Epidermal growth factor 8681176 IL-1RA Interleukin-1 receptor-alpha 2689 3640 PDGF-BBPlatelet-derived growth 980 1327 factor subunit B-hetero- dimer SCF SCFcomplex 29 40 VEGF-A Vascular endothelial growth 585 792 factor-A

In other embodiments, an alternative composition may be produced byvarying particular steps in aliquoting mononuclear cells and/or by useof the inspection and acceptance or rejection process, to produceeffective concentrations within the composition of particular cytokines,as listed in Table 11 below.

TABLE 11 Minimum Maximum Cytokine Concentration Concentration SymbolCytokine (picograms/ml) (picograms/ml) EGF Epidermal growth factor 9191124 IL-1RA Interleukin-1 receptor-alpha 2848 3481 PDGF-BBPlatelet-derived growth 1038 1269 factor subunit B-hetero- dimer SCF SCFcomplex 30 38 VEGF-A Vascular endothelial growth 619 758 factor-A

In other embodiments, an alternative composition may be produced byvarying particular steps in aliquoting mononuclear cells and/or by useof the inspection and acceptance or rejection process, to produceeffective concentrations within the composition of particular cytokines,as listed in Table 12 below.

TABLE 12 Minimum Maximum Cytokine Concentration Concentration SymbolCytokine (picograms/ml) (picograms/ml) EGF Epidermal growth factor 9701073 IL-1RA Interleukin-1 receptor-alpha 3006 3323 PDGF-BBPlatelet-derived growth 1095 1212 factor subunit B-hetero- dimer SCF SCFcomplex 32 36 VEGF-A Vascular endothelial growth 653 723 factor-A

In other embodiments, an alternative composition may be produced byvarying particular steps in aliquoting mononuclear cells and/or by useof the inspection and acceptance or rejection process, to produceeffective concentrations within the composition of particular cytokines,as listed in Table 13 below.

TABLE 13 Minimum Maximum Concen- Concen- tration tration Cytokine (pico-(pico- Symbol Cytokine grams/ml) grams/ml) EGF Epidermal growth factor715 1329 GM-CSF Granulocyte-macrophage colony 7 15 stimulating factorIL-1RA Interleukin-1 receptor-alpha 2215 4114 IL-4 Interleukin-4 17 33IL-8 Interleukin-8 4504 8366 PDGF-BB Platelet-derived growth factor 8071500 subunit B-heterodimer RANTES Chemokine (C-C motif) ligand 5 32776087 SCF SCF complex 23 45 SDF-1alpha Stromal cell-derived factor-1alpha 1114 2071 VEGF-A Vascular endothelial growth 481 895 factor-A

In other embodiments, an alternative composition may be produced byvarying particular steps in aliquoting mononuclear cells and/or by useof the inspection and acceptance or rejection process, to produceeffective concentrations within the composition of particular cytokines,as listed in Table 14 below.

TABLE 14 Minimum Maximum Concen- Concen- tration tration Cytokine (pico-(pico- Symbol Cytokine grams/ml) grams/ml) EGF Epidermal growth factor766 1278 GM-CSF Granulocyte-macrophage colony 8 15 stimulating factorIL-1RA Interleukin-1 receptor-alpha 2373 3956 IL-4 Interleukin-4 18 32IL-8 Interleukin-8 4826 8044 PDGF-BB Platelet-derived growth factor 8651442 subunit B-heterodimer RANTES Chemokine (C-C motif) ligand 5 35115853 SCF SCF complex 25 43 SDF-1alpha Stromal cell-derived factor-1alpha 1194 1991 VEGF-A Vascular endothelial growth 516 861 factor-A

In other embodiments, an alternative composition may be produced byvarying particular steps in aliquoting mononuclear cells and/or by useof the inspection and acceptance or rejection process, to produceeffective concentrations within the composition of particular cytokines,as listed in Table 15 below.

TABLE 15 Minimum Maximum Concen- Concen- tration tration Cytokine (pico-(pico- Symbol Cytokine grams/ml) grams/ml) EGF Epidermal growth factor817 1227 GM-CSF Granulocyte-macrophage colony 9 14 stimulating factorIL-1RA Interleukin-1 receptor-alpha 2531 3798 IL-4 Interleukin-4 19 30IL-8 Interleukin-8 5147 7722 PDGF-BB Platelet-derived growth factor 9221385 subunit B-heterodimer RANTES Chemokine (C-C motif) ligand 5 37455619 SCF SCF complex 27 42 SDF-1alpha Stromal cell-derived factor-1alpha 1273 1911 VEGF-A Vascular endothelial growth 550 826 factor-A

In other embodiments, an alternative composition may be produced byvarying particular steps in aliquoting mononuclear cells and/or by useof the inspection and acceptance or rejection process, to produceeffective concentrations within the composition of particular cytokines,as listed in Table 16 below.

TABLE 16 Minimum Maximum Concen- Concen- tration tration Cytokine (pico-(pico- Symbol Cytokine grams/ml) grams/ml) EGF Epidermal growth factor868 1176 GM-CSF Granulocyte-macrophage colony 9 13 stimulating factorIL-1RA Interleukin-1 receptor-alpha 2689 3640 IL-4 Interleukin-4 21 29IL-8 Interleukin-8 5469 7401 PDGF-BB Platelet-derived growth factor 9801327 subunit B-heterodimer RANTES Chemokine (C-C motif) ligand 5 39795385 SCF SCF complex 29 40 SDF-1alpha Stromal cell-derived factor-1alpha 1353 1832 VEGF-A Vascular endothelial growth 585 792 factor-A

In other embodiments, an alternative composition may be produced byvarying particular steps in aliquoting mononuclear cells and/or by useof the inspection and acceptance or rejection process, to produceeffective concentrations within the composition of particular cytokines,as listed in Table 17 below.

TABLE 17 Minimum Maximum Concen- Concen- tration tration Cytokine (pico-(pico- Symbol Cytokine grams/ml) grams/ml) EGF Epidermal growth factor919 1124 GM-CSF Granulocyte-macrophage colony 10 13 stimulating factorIL-1RA Interleukin-1 receptor-alpha 2848 3481 IL-4 Interleukin-4 22 28IL-8 Interleukin-8 5791 7079 PDGF-BB Platelet-derived growth factor 10381269 subunit B-heterodimer RANTES Chemokine (C-C motif) ligand 5 42135151 SCF SCF complex 30 38 SDF-1alpha Stromal cell-derived factor-1alpha 1433 1752 VEGF-A Vascular endothelial growth 619 758 factor-A

In other embodiments, an alternative composition may be produced byvarying particular steps in aliquoting mononuclear cells and/or by useof the inspection and acceptance or rejection process, to produceeffective concentrations within the composition of particular cytokines,as listed in Table 18 below.

TABLE 18 Minimum Maximum Concen- Concen- tration tration Cytokine (pico-(pico- Symbol Cytokine grams/ml) grams/ml) EGF Epidermal growth factor970 1073 GM-CSF Granulocyte-macrophage colony 10 12 stimulating factorIL-1RA Interleukin-1 receptor-alpha 3006 3323 IL-4 Interleukin-4 23 27IL-8 Interleukin-8 6113 6757 PDGF-BB Platelet-derived growth factor 10951212 subunit B-heterodimer RANTES Chemokine (C-C motif) ligand 5 44474916 SCF SCF complex 32 36 SDF-1alpha Stromal cell-derived factor-1alpha 1512 1672 VEGF-A Vascular endothelial growth 653 723 factor-A

FIGS. 26A-28B illustrate compositions comprising embodiments describedherein in use in medical treatment methods. FIGS. 26A-26C illustrate aprocedure for spinal fusion using a composition of an embodimentdescribed herein. A spine 52 of a patient includes two adjacentvertebrae 54, 56 which are to be fused. It may be desired to fuse two ormore vertebrae to treat a number of different ailments, including, butnot limited to: scoliosis, spondylolisthesis, hyperkyphosis,hyperlordosis (or hypolordosis/flat-back syndrome, hypokyphosis),degenerative disk disease (DDD), spinal stenosis, trauma, includingvertebral fracture, tumor, revision surgery, infection, pseudoarthrosis,herniated disk, spondylolysis, mechanical instability, facet syndrome,chronic back pain, and radiating leg pain. In FIG. 26A, a patient'svertebrae 54, 56 are at least partially exposed and an intervertebraldisk 58 is removed. In some cases, the disk 58 may already have beenremoved or may be missing or damaged, and thus not require removal. Insome cases, at least a portion of the disk may be purposely left inplace. Though in some cases it is optional, in FIG. 26B, a load-bearingdevice 60 is placed between the first vertebrae 54 and the secondvertebrae 56. The load-bearing device 60 may include a fusion cage orinterbody device. In FIG. 26C, a biological composition 62, such as anyembodiment of those described herein, is placed between the vertebrae54, 56 and, when applicable, around or adjacent to the load-bearingdevice 60, including within any cavities in the interbody device, shouldthere be any. The biological composition 62, may also be placed near orat posterior-lateral gutters. Procedures may include posterior lumbarinterbody fusion (PLIF), transforaminal lumbar interbody fusion (TLIF),anterior lumbar interbody fusion (ALIF), extreme lateral interbodyfusion (XLIF), direct lateral interbody fusion (DLIF), or axial lumbarinterbody fusion (AliaLIF). An appropriate amount of the biologicalcomposition 62 is placed so that it is effective in aiding the fusion ofthe two vertebrae 54, 56. In some cases the patient may be skeletallymature. In other cases, the patient may be skeletally immature.

In some cases, an effective amount of the biological composition 62 topromote, aid, or accelerate fusion of the first vertebra 54 to thesecond vertebra 56 may comprise between about 1% and 50% of a volume tobe filled, or about 3% to 25%, or about 5% to 20%, or about 10%. Theremainder of the volume to be filled may be filled with bone graft, orother materials, or left at least partially unfilled. For example, in adisc space of about 10 ml, about 0.5 ml to 2.0 ml of the biologicalcomposition 62 may be inserted, or in a disc space of about 5 ml, about0.25 ml to about 1.0 ml of the biological composition 62 may beinserted. The biological composition 62 may be applied substantially toa particular location within the volume to be filled, or alternativelymay be applied in diffuse locations within the volume to be filled. Insome embodiments, other materials may be added to the biologicalcomposition 62, including synthetic bone substitutes such as betatricalcium phosphate, or actual pieces of bone, including ground bone.

FIGS. 27A-27B illustrate a procedure for fusion using a composition ofan embodiment described herein. A segment 72 of the skeletal system of apatient includes a first bone portion 74 and a second bone portion 76which are to be fused. It may be desired to fuse two or more boneportions to treat a number of different ailments, including, but notlimited to: trauma, including fracture, tumor, arthritis, ankylosis,deformity, congenital defects, bone length discrepancy, revisionsurgery, infection, osteonecrosis, osteoporosis, pseudoarthrosis,non-union, delayed-union, and pain. The bone portions 74, 76 may both bewithin a particular portion of the spine (cervical, thoracic, lumbar,sacral) or may be within two different portions (thoracic to lumbar,lumbar to sacral, etc.). In FIG. 27A, the first bone portion 74 and thesecond bone portion 76 are at least partially exposed. Surfaces in oneor each of the first and second bone portions 74, 76 may then beprepared, for example by cutting (osteotomy) or roughening. In somecases, a load-bearing device 75, such as a plate, rod, nail, screw,hook, or pin may be used to hold the first bone portion 74 and thesecond bone portion 76 in relation to one another. In FIG. 27B, abiological composition 78, such as any embodiment of those describedherein, is placed between the first and second bone portions 74, 76 and,when applicable, around or adjacent to the load-bearing device 75. Anappropriate amount of the biological composition 78 is placed so that itis effective in aiding the fusion of first and second bone portions 74,76. Any of the bones of the skeletal system may be fused while utilizingthe biological composition 78. In some cases, the patient may beskeletally mature. In other cases, the patient may be skeletallyimmature.

In some cases, an effective amount of the biological composition 78 topromote, aid, or accelerate fusion of the first bone portion 74 to thesecond bone portion 76 may comprise between about 1% and 50% of a volumeto be filled, or about 3% to 25%, or about 5% to 20%, or about 10%. Theremainder of the volume to be filled may be filled with bone graft, orother materials, or left at least partially unfilled. The biologicalcomposition 78 may be applied substantially to a particular locationwithin the volume to be filled, or alternatively may be applied indiffuse locations within the volume to be filled. In some embodiments,other materials may be added to the biological composition 78, includingsynthetic bone substitutes such as beta tricalcium phosphate, or actualpieces of bone, including ground bone.

FIGS. 28A-28B illustrate a procedure for healing or growth augmentationof soft tissue 84 in a region 82 of a patient. A biological composition86, such as any embodiment of those described herein, is placed within,near, or adjacent the soft tissue 84. In alternative procedures,partially or fully hard tissue may also or instead be treated with thebiological composition 86. In some cases, the patient to be treated hasat least some cancerous cells, and the biological composition 86 isplaced within, near, or adjacent the cancerous cells. In some cases, thepatient to be treated had at least some cancerous cells removed, and thebiological composition 86 is placed within, near, or adjacent theprevious location (former site) of the cancerous cells. In cancerpatients or post-cancer patients, the cancer may include, but is notlimited to: lymphatic cancer, including lymph vessel tumors, soft tissuesarcoma, fat tissue tumors, muscle tissue tumors, peripheral nervetumors, fibrous tissue tumors, joint tissue tumors, and blood vesseltumors. In some cases, the biological composition may be used forvascular reconstruction (e.g., blood vessels). The patients treated mayinclude adult or pediatric patients. In some cases, an effective amountof the biological composition 86 to promote healing or growthaugmentation of the soft tissue 84 may comprise at least about 0.25 ml,or between about 0.25 ml and about 5 ml, or between about 0.25 ml andabout 2 ml.

Other sources besides umbilical cord blood are possible for thecompositions described herein, including, but not limited to amnioticfluid, peripheral blood, umbilical cord tissue, bone marrow, adiposetissue, and central nervous system (CNS) fluid.

In addition to previously mentioned procedures, other procedures may beperformed incorporating the compositions and methods of manufacture ofthe embodiments described herein, including, but not limited toprocedures for the treatment of diabetes mellitus and/or its relatedsymptoms, procedures for the treatment of diabetic neuropathy,procedures for the treatment of diabetic ulcers, procedures for thetreatment of epidermal, dermal, or sub-dermal diabetic ulcers,procedures for pain management for joints, including joints havingchronic pain, procedures for the treatment of tendonitis, procedures forthe treatment of torn cartilage, procedures for the treatment of tendonlaxity, such as tendon laxity in joints, including but not limited to,shoulder joints, ankle joints, knee joints, hip joints, and elbowjoints, procedures for the treatment of leukemia, procedures for thetreatment of lymphoma, procedures for the treatment of myeloma,procedures for the treatment of other cancers, procedures for otherepidermal, dermal, or sub-dermal disorders or illnesses, or proceduresrelated to damage to neurons, including Parkinson's disease and braintumors. Procedures may be performed incorporating the compositions andmethods of manufacture of the embodiments described herein to reducetumor size. The compositions may in some embodiments, be added in othermanners, including epidural application, transthecal application,intravenous (IV) application, or direct site application.

While embodiments have been shown and described, various modificationsmay be made without departing from the scope of the inventive conceptsdisclosed herein.

The ranges disclosed herein also encompass any and all overlap,sub-ranges, and combinations thereof. Language such as “up to,” “atleast,” “greater than,” “less than,” “between,” and the like includesthe number recited. Numbers preceded by a term such as “approximately”,“about”, and “substantially” as used herein include the recited numbers(e.g., about 10%=10%), and also represent an amount close to the statedamount that still performs a desired function or achieves a desiredresult. For example, the terms “approximately”, “about”, and“substantially” may refer to an amount that is within less than 10% of,within less than 5% of, within less than 1% of, within less than 0.1%of, and within less than 0.01% of the stated amount.

What is claimed is:
 1. A composition for influencing biological growthcomprising: a low pH fluid base, wherein the low pH fluid base comprisesdextrose; mononuclear cells obtained from human umbilical cord blood;VEGF-A, at a concentration within the composition of between 481picograms per ml and 895 picograms per ml; PDGF-BB, at a concentrationwithin the composition of between 807 picograms per ml and 1500picograms per ml; EGF, at a concentration within the composition ofbetween 715 picograms per ml and 1329 picograms per ml; SCF, at aconcentration within the composition of between 23 picograms per ml and45 picograms per ml; and IL-1RA, at a concentration within thecomposition of between 2215 picograms per ml and 4114 picograms per ml,wherein the composition is configured for implantation within a humansubject.
 2. The composition of claim 1, wherein: the concentrationwithin the composition of VEGF-A is between 516 picograms per ml and 861picograms per ml; the concentration within the composition of PDGF-BB isbetween 865 picograms per ml and 1442 picograms per ml; theconcentration within the composition of EGF is between 766 picograms perml and 1278 picograms per ml; the concentration within the compositionof SCF is between 25 picograms per ml and 43 picograms per ml; and theconcentration within the composition of IL-1RA is between 2373 picogramsper ml and 3956 picograms per ml.
 3. The composition of claim 1,wherein: the concentration within the composition of VEGF-A is between550 picograms per ml and 826 picograms per ml; the concentration withinthe composition of PDGF-BB is between 922 picograms per ml and 1385picograms per ml; the concentration within the composition of EGF isbetween 817 picograms per ml and 1227 picograms per ml; theconcentration within the composition of SCF is between 27 picograms perml and 42 picograms per ml; and the concentration within the compositionof IL-1RA is between 2531 picograms per ml and 3798 picograms per ml. 4.The composition of claim 1, wherein: the concentration within thecomposition of VEGF-A is between 585 picograms per ml and 792 picogramsper ml; the concentration within the composition of PDGF-BB is between980 picograms per ml and 1327 picograms per ml; the concentration withinthe composition of EGF is between 868 picograms per ml and 1176picograms per ml; the concentration within the composition of SCF isbetween 29 picograms per ml and 40 picograms per ml; and theconcentration within the composition of IL-1RA is between 2689 picogramsper ml and 3640 picograms per ml.
 5. The composition of claim 1,wherein: the concentration within the composition of VEGF-A is between619 picograms per ml and 758 picograms per ml; the concentration withinthe composition of PDGF-BB is between 1038 picograms per ml and 1269picograms per ml; the concentration within the composition of EGF isbetween 919 picograms per ml and 1124 picograms per ml; theconcentration within the composition of SCF is between 30 picograms perml and 38 picograms per ml; and the concentration within the compositionof IL-1RA is between 2848 picograms per ml and 3481 picograms per ml. 6.The composition of claim 1, wherein: the concentration within thecomposition of VEGF-A is between 653 picograms per ml and 723 picogramsper ml; the concentration within the composition of PDGF-BB is between1095 picograms per ml and 1212 picograms per ml; the concentrationwithin the composition of EGF is between 970 picograms per ml and 1073picograms per ml; the concentration within the composition of SCF isbetween 32 picograms per ml and 36 picograms per ml; and theconcentration within the composition of IL-1RA is between 3006 picogramsper ml and 3323 picograms per ml, and wherein the composition furthercomprises: GM-CSF having a concentration within the composition ofbetween 10 picograms per ml and 12 picograms per ml; IL-4 having aconcentration within the composition of between 23 picograms per ml and27 picograms per ml; IL-8 having a concentration within the compositionof between 6113 picograms per ml and 6757 picograms per ml; SDF-1αhaving a concentration within the composition of between 1512 picogramsper ml and 1672 picograms per ml; and RANTES having a concentrationwithin the composition of between 4447 picograms per ml and 4916picograms per ml.
 7. A composition for influencing biological growthcomprising: a low pH fluid base, wherein the low pH fluid base comprisesdextrose; mononuclear cells obtained from human umbilical cord blood;and wherein the composition includes the following cytokines with theirconcentration within the composition: BDNF at between 492 picograms perml and 915 picograms per ml, bNGF at between 14 picograms per ml and 26picograms per ml, EGF at between 715 picograms per ml and 1329 picogramsper ml, Eotaxin at between 37 picograms per ml and 69 picograms per ml,FGF-2 at between 28 picograms per ml and 53 picograms per ml, GM-CSF atbetween 7 picograms per ml and 15 picograms per ml, Gro-α at between2974 picograms per ml and 5524 picograms per ml, HGF at between 352picograms per ml and 656 picograms per ml, IFN-α at between 253picograms per ml and 472 picograms per ml, IFN-γ at between 33 picogramsper ml and 63 picograms per ml, IL-10 at between 67 picograms per ml and126 picograms per ml, IL-12p70 at between 9 picograms per ml and 19picograms per ml, IL-13 at between 11 picograms per ml and 22 picogramsper ml, IL-15 at between 30 picograms per ml and 57 picograms per ml,IL-17A at between 19 picograms per ml and 37 picograms per ml, IL-18 atbetween 78 picograms per ml and 146 picograms per ml, IL-1α at between712 picograms per ml and 1324 picograms per ml, IL-1β at between 14picograms per ml and 28 picograms per ml, IL-1RA at between 2215picograms per ml and 4114 picograms per ml, IL-2 at between 16 picogramsper ml and 31 picograms per ml, IL-21 at between 370 picograms per mland 689 picograms per ml, IL-22 at between 16 picograms per ml and 32picograms per ml, IL-23 at between 201 picograms per ml and 376picograms per ml, IL-27 at between 21 picograms per ml and 40 picogramsper ml, IL-31 at between 11 picograms per ml and 22 picograms per ml,IL-4 at between 17 picograms per ml and 33 picograms per ml, IL-5 atbetween 15 picograms per ml and 28 picograms per ml, IL-6 at between 50picograms per ml and 95 picograms per ml, IL-7 at between 26 picogramsper ml and 50 picograms per ml, IL-8 at between 4504 picograms per mland 8366 picograms per ml, IL-9 at between 131 picograms per ml and 245picograms per ml, IP-10 at between 469 picograms per ml and 873picograms per ml, LIF at between 41 picograms per ml and 77 picogramsper ml, MCP-1 at between 14,480 picograms per ml and 26,893 picogramsper ml, MIP-1α at between 303 picograms per ml and 565 picograms per ml,MIP-1β at between 375 picograms per ml and 698 picograms per ml, PDGF-BBat between 807 picograms per ml and 1500 picograms per ml, PIGF-1 atbetween 743 picograms per ml and 1381 picograms per ml, RANTES atbetween 3277 picograms per ml and 6087 picograms per ml, SCF at between23 picograms per ml and 45 picograms per ml, SDF-1α at between 1114picograms per ml and 2071 picograms per ml, TNF-α at between 42picograms per ml and 80 picograms per ml, TNF-β at between 12 picogramsper ml and 25 picograms per ml, VEGF-A at between 481 picograms per mland 895 picograms per ml, and VEGF-D at between 46 picograms per ml and88 picograms per ml, wherein the composition is configured forimplantation within a human subject.
 8. The composition of claim 7,wherein the concentrations of the cytokines are: BDNF at between 527picograms per ml and 880 picograms per ml, bNGF at between 15 picogramsper ml and 25 picograms per ml, EGF at between 766 picograms per ml and1278 picograms per ml, Eotaxin at between 39 picograms per ml and 67picograms per ml, FGF-2 at between 30 picograms per ml and 51 picogramsper ml, GM-CSF at between 8 picograms per ml and 15 picograms per ml,Gro-α at between 3186 picograms per ml and 5312 picograms per ml, HGF atbetween 378 picograms per ml and 631 picograms per ml, IFN-α at between271 picograms per ml and 454 picograms per ml, IFN-γ at between 35picograms per ml and 60 picograms per ml, IL-10 at between 72 picogramsper ml and 121 picograms per ml, IL-12p70 at between 10 picograms per mland 18 picograms per ml, IL-13 at between 12 picograms per ml and 21picograms per ml, IL-15 at between 32 picograms per ml and 55 picogramsper ml, IL-17A at between 21 picograms per ml and 35 picograms per ml,IL-18 at between 83 picograms per ml and 140 picograms per ml, IL-1α atbetween 763 picograms per ml and 1273 picograms per ml, IL-1β at between15 picograms per ml and 27 picograms per ml, IL-1RA at between 2373picograms per ml and 3956 picograms per ml, IL-2 at between 17 picogramsper ml and 30 picograms per ml, IL-21 at between 396 picograms per mland 662 picograms per ml, IL-22 at between 17 picograms per ml and 30picograms per ml, IL-23 at between 216 picograms per ml and 361picograms per ml, IL-27 at between 22 picograms per ml and 38 picogramsper ml, IL-31 at between 12 picograms per ml and 21 picograms per ml,IL-4 at between 18 picograms per ml and 32 picograms per ml, IL-5 atbetween 16 picograms per ml and 27 picograms per ml, IL-6 at between 54picograms per ml and 91 picograms per ml, IL-7 at between 28 picogramsper ml and 48 picograms per ml, IL-8 at between 4826 picograms per mland 8044 picograms per ml, IL-9 at between 141 picograms per ml and 236picograms per ml, IP-10 at between 503 picograms per ml and 840picograms per ml, LIF at between 44 picograms per ml and 74 picogramsper ml, MCP-1 at between 15,514 picograms per ml and 25,859 picogramsper ml, MIP-1α at between 325 picograms per ml and 543 picograms per ml,MIP-1β at between 402 picograms per ml and 671 picograms per ml, PDGF-BBat between 865 picograms per ml and 1442 picograms per ml, PIGF-1 atbetween 796 picograms per ml and 1328 picograms per ml, RANTES atbetween 3511 picograms per ml and 5853 picograms per ml, SCF at between25 picograms per ml and 43 picograms per ml, SDF-1α at between 1194picograms per ml and 1991 picograms per ml, TNF-α at between 45picograms per ml and 77 picograms per ml, TNF-β at between 13 picogramsper ml and 24 picograms per ml, VEGF-A at between 516 picograms per mland 861 picograms per ml, and VEGF-D at between 50 picograms per ml and84 picograms per ml.
 9. The composition of claim 7, wherein theconcentrations of the cytokines are: BDNF at between 562 picograms perml and 844 picograms per ml, bNGF at between 16 picograms per ml and 24picograms per ml, EGF at between 817 picograms per ml and 1227 picogramsper ml, Eotaxin at between 42 picograms per ml and 64 picograms per ml,FGF-2 at between 32 picograms per ml and 49 picograms per ml, GM-CSF atbetween 9 picograms per ml and 14 picograms per ml, Gro-α at between3399 picograms per ml and 5099 picograms per ml, HGF at between 403picograms per ml and 605 picograms per ml, IFN-α at between 290picograms per ml and 436 picograms per ml, IFN-γ at between 38 picogramsper ml and 57 picograms per ml, IL-10 at between 77 picograms per ml and117 picograms per ml, IL-12p70 at between 11 picograms per ml and 18picograms per ml, IL-13 at between 13 picograms per ml and 20 picogramsper ml, IL-15 at between 34 picograms per ml and 53 picograms per ml,IL-17A at between 22 picograms per ml and 34 picograms per ml, IL-18 atbetween 89 picograms per ml and 135 picograms per ml, IL-1α at between814 picograms per ml and 1222 picograms per ml, IL-1β at between 17picograms per ml and 26 picograms per ml, IL-1RA at between 2531picograms per ml and 3798 picograms per ml, IL-2 at between 18 picogramsper ml and 29 picograms per ml, IL-21 at between 423 picograms per mland 636 picograms per ml, IL-22 at between 19 picograms per ml and 29picograms per ml, IL-23 at between 230 picograms per ml and 347picograms per ml, IL-27 at between 24 picograms per ml and 37 picogramsper ml, IL-31 at between 13 picograms per ml and 20 picograms per ml,IL-4 at between 19 picograms per ml and 30 picograms per ml, IL-5 atbetween 17 picograms per ml and 26 picograms per ml, IL-6 at between 58picograms per ml and 88 picograms per ml, IL-7 at between 30 picogramsper ml and 46 picograms per ml, IL-8 at between 5147 picograms per mland 7722 picograms per ml, IL-9 at between 150 picograms per ml and 226picograms per ml, IP-10 at between 537 picograms per ml and 806picograms per ml, LIF at between 47 picograms per ml and 71 picogramsper ml, MCP-1 at between 16,549 picograms per ml and 24,824 picogramsper ml, MIP-1α at between 347 picograms per ml and 522 picograms per ml,MIP-1β at between 429 picograms per ml and 644 picograms per ml, PDGF-BBat between 922 picograms per ml and 1385 picograms per ml, PIGF-1 atbetween 849 picograms per ml and 1275 picograms per ml, RANTES atbetween 3745 picograms per ml and 5619 picograms per ml, SCF at between27 picograms per ml and 42 picograms per ml, SDF-1α at between 1273picograms per ml and 1911 picograms per ml, TNF-α at between 49picograms per ml and 74 picograms per ml, TNF-β at between 14 picogramsper ml and 23 picograms per ml, VEGF-A at between 550 picograms per mland 826 picograms per ml, and VEGF-D at between 53 picograms per ml and81 picograms per ml.
 10. The composition of claim 7, wherein theconcentrations of the cytokines are: BDNF at between 597 picograms perml and 809 picograms per ml, bNGF at between 17 picograms per ml and 23picograms per ml, EGF at between 868 picograms per ml and 1176 picogramsper ml, Eotaxin at between 45 picograms per ml and 61 picograms per ml,FGF-2 at between 34 picograms per ml and 47 picograms per ml, GM-CSF atbetween 9 picograms per ml and 13 picograms per ml, Gro-α at between3611 picograms per ml and 4887 picograms per ml, HGF at between 428picograms per ml and 580 picograms per ml, IFN-α at between 308picograms per ml and 418 picograms per ml, IFN-γ at between 40 picogramsper ml and 55 picograms per ml, IL-10 at between 82 picograms per ml and112 picograms per ml, IL-12p70 at between 12 picograms per ml and 17picograms per ml, IL-13 at between 13 picograms per ml and 19 picogramsper ml, IL-15 at between 36 picograms per ml and 51 picograms per ml,IL-17A at between 23 picograms per ml and 33 picograms per ml, IL-18 atbetween 95 picograms per ml and 129 picograms per ml, IL-1α at between865 picograms per ml and 1171 picograms per ml, IL-1α at between 18picograms per ml and 25 picograms per ml, IL-1RA at between 2689picograms per ml and 3640 picograms per ml, IL-2 at between 20 picogramsper ml and 28 picograms per ml, IL-21 at between 449 picograms per mland 609 picograms per ml, IL-22 at between 20 picograms per ml and 28picograms per ml, IL-23 at between 245 picograms per ml and 332picograms per ml, IL-27 at between 25 picograms per ml and 35 picogramsper ml, IL-31 at between 13 picograms per ml and 19 picograms per ml,IL-4 at between 21 picograms per ml and 29 picograms per ml, IL-5 atbetween 18 picograms per ml and 25 picograms per ml, IL-6 at between 61picograms per ml and 84 picograms per ml, IL-7 at between 32 picogramsper ml and 44 picograms per ml, IL-8 at between 5469 picograms per mland 7401 picograms per ml, IL-9 at between 160 picograms per ml and 217picograms per ml, IP-10 at between 570 picograms per ml and 772picograms per ml, LIF at between 50 picograms per ml and 68 picogramsper ml, MCP-1 at between 17,583 picograms per ml and 23,790 picogramsper ml, MIP-1α at between 369 picograms per ml and 500 picograms per ml,MIP-1β at between 455 picograms per ml and 617 picograms per ml, PDGF-BBat between 980 picograms per ml and 1327 picograms per ml, PIGF-1 atbetween 902 picograms per ml and 1222 picograms per ml, RANTES atbetween 3979 picograms per ml and 5385 picograms per ml, SCF at between29 picograms per ml and 40 picograms per ml, SDF-1α at between 1353picograms per ml and 1832 picograms per ml, TNF-α at between 52picograms per ml and 71 picograms per ml, TNF-β at between 15 picogramsper ml and 22 picograms per ml, VEGF-A at between 585 picograms per mland 792 picograms per ml, and VEGF-D at between 56 picograms per ml and78 picograms per ml.
 11. The composition of claim 7, wherein theconcentrations of the cytokines are: BDNF at between 632 picograms perml and 774 picograms per ml, bNGF at between 18 picograms per ml and 22picograms per ml, EGF at between 919 picograms per ml and 1124 picogramsper ml, Eotaxin at between 47 picograms per ml and 59 picograms per ml,FGF-2 at between 36 picograms per ml and 45 picograms per ml, GM-CSF atbetween 10 picograms per ml and 13 picograms per ml, Gro-α at between3823 picograms per ml and 4674 picograms per ml, HGF at between 453picograms per ml and 555 picograms per ml, IFN-α at between 326picograms per ml and 399 picograms per ml, IFN-γ at between 42 picogramsper ml and 53 picograms per ml, IL-10 at between 87 picograms per ml and107 picograms per ml, IL-12p70 at between 12 picograms per ml and 16picograms per ml, IL-13 at between 14 picograms per ml and 19 picogramsper ml, IL-15 at between 39 picograms per ml and 48 picograms per ml,IL-17A at between 25 picograms per ml and 31 picograms per ml, IL-18 atbetween 100 picograms per ml and 124 picograms per ml, IL-1α at between916 picograms per ml and 1121 picograms per ml, IL-1β at between 19picograms per ml and 24 picograms per ml, IL-1RA at between 2848picograms per ml and 3481 picograms per ml, IL-2 at between 21 picogramsper ml and 26 picograms per ml, IL-21 at between 476 picograms per mland 583 picograms per ml, IL-22 at between 21 picograms per ml and 27picograms per ml, IL-23 at between 259 picograms per ml and 318picograms per ml, IL-27 at between 27 picograms per ml and 34 picogramsper ml, IL-31 at between 14 picograms per ml and 18 picograms per ml,IL-4 at between 22 picograms per ml and 28 picograms per ml, IL-5 atbetween 19 picograms per ml and 24 picograms per ml, IL-6 at between 65picograms per ml and 81 picograms per ml, IL-7 at between 34 picogramsper ml and 42 picograms per ml, IL-8 at between 5791 picograms per mland 7079 picograms per ml, IL-9 at between 169 picograms per ml and 208picograms per ml, IP-10 at between 604 picograms per ml and 739picograms per ml, LIF at between 53 picograms per ml and 66 picogramsper ml, MCP-1 at between 18,617 picograms per ml and 22,756 picogramsper ml, MIP-1α at between 390 picograms per ml and 478 picograms per ml,MIP-1β at between 482 picograms per ml and 590 picograms per ml, PDGF-BBat between 1038 picograms per ml and 1269 picograms per ml, PIGF-1 atbetween 955 picograms per ml and 1169 picograms per ml, RANTES atbetween 4213 picograms per ml and 5151 picograms per ml, SCF at between30 picograms per ml and 38 picograms per ml, SDF-1α at between 1433picograms per ml and 1752 picograms per ml, TNF-α at between 55picograms per ml and 68 picograms per ml, TNF-β at between 16 picogramsper ml and 21 picograms per ml, VEGF-A at between 619 picograms per mland 758 picograms per ml, and VEGF-D at between 60 picograms per ml and74 picograms per ml.
 12. The composition of claim 7, wherein theconcentrations of the cytokines are: BDNF at between 668 picograms perml and 739 picograms per ml, bNGF at between 19 picograms per ml and 21picograms per ml, EGF at between 970 picograms per ml and 1073 picogramsper ml, Eotaxin at between 50 picograms per ml and 56 picograms per ml,FGF-2 at between 38 picograms per ml and 43 picograms per ml, GM-CSF atbetween 10 picograms per ml and 12 picograms per ml, Gro-α at between4036 picograms per ml and 4462 picograms per ml, HGF at between 478picograms per ml and 530 picograms per ml, IFN-α at between 344picograms per ml and 381 picograms per ml, IFN-γ at between 45 picogramsper ml and 50 picograms per ml, IL-10 at between 91 picograms per ml and102 picograms per ml, IL-12p70 at between 13 picograms per ml and 15picograms per ml, IL-13 at between 15 picograms per ml and 18 picogramsper ml, IL-15 at between 41 picograms per ml and 46 picograms per ml,IL-17A at between 26 picograms per ml and 30 picograms per ml, IL-18 atbetween 106 picograms per ml and 118 picograms per ml, IL-1α at between967 picograms per ml and 1070 picograms per ml, IL-1β at between 20picograms per ml and 23 picograms per ml, IL-1RA at between 3006picograms per ml and 3323 picograms per ml, IL-2 at between 22 picogramsper ml and 25 picograms per ml, IL-21 at between 502 picograms per mland 556 picograms per ml, IL-22 at between 22 picograms per ml and 26picograms per ml, IL-23 at between 274 picograms per ml and 303picograms per ml, IL-27 at between 28 picograms per ml and 32 picogramsper ml, IL-31 at between 15 picograms per ml and 18 picograms per ml,IL-4 at between 23 picograms per ml and 27 picograms per ml, IL-5 atbetween 20 picograms per ml and 23 picograms per ml, IL-6 at between 69picograms per ml and 77 picograms per ml, IL-7 at between 36 picogramsper ml and 40 picograms per ml, IL-8 at between 6113 picograms per mland 6757 picograms per ml, IL-9 at between 178 picograms per ml and 198picograms per ml, IP-10 at between 637 picograms per ml and 705picograms per ml, LIF at between 56 picograms per ml and 63 picogramsper ml, MCP-1 at between 19,652 picograms per ml and 21,721 picogramsper ml, MIP-1α at between 412 picograms per ml and 456 picograms per ml,MIP-1β at between 509 picograms per ml and 564 picograms per ml, PDGF-BBat between 1095 picograms per ml and 1212 picograms per ml, PIGF-1 atbetween 1009 picograms per ml and 1116 picograms per ml, RANTES atbetween 4447 picograms per ml and 4916 picograms per ml, SCF at between32 picograms per ml and 36 picograms per ml, SDF-1α at between 1512picograms per ml and 1672 picograms per ml, TNF-α at between 58picograms per ml and 65 picograms per ml, TNF-β at between 17 picogramsper ml and 20 picograms per ml, VEGF-A at between 653 picograms per mland 723 picograms per ml, and VEGF-D at between 63 picograms per ml and71 picograms per ml.
 13. The composition of claim 1, wherein the low pHfluid base further comprises dextran.
 14. The composition of claim 1,further comprising albumin.
 15. The composition of claim 7, wherein thelow pH fluid base further comprises dextran.
 16. The composition ofclaim 7, further comprising albumin.